GM-CSF and IL-4 conjugates, compositions, and methods related thereto

ABSTRACT

In certain embodiments, this disclosure relates to conjugates comprising a polypeptide of GM-CSF and a polypeptide IL-4. Typically, the GM-CSF and IL-4 are connected by a linker, e.g., polypeptide. In certain embodiments, the disclosure relates to isolated nucleic acids encoding these polypeptide conjugates, vectors comprising nucleic acid encoding polypeptide conjugates, and protein expression systems comprising these vectors such as infectious viral particles and host cells comprising such nucleic acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/436,491filed Apr. 17, 2015, which is the National Stage of InternationalApplication Number PCT/US2013/066261 filed Oct. 23, 2013, which claimsthe benefit of priority to U.S. Provisional Application No. 61/717,129filed Oct. 23, 2012. The entirety of each of these applications ishereby incorporated by reference for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 12066USDIV_ST25.txt. The text file is 12 KB, wascreated on Apr. 19, 2017, and is being submitted electronically viaEFS-Web.

BACKGROUND

Cancer is thought to occur as a result of an immune system that is notproperly removing uncontrolled proliferating cancer cells. Stimulatingthe immune system to recognize and eliminate cancerous cells has becomea promising strategy for therapeutic treatments. Proleukin®(aldesleukin) contains a recombinant human Interleukin 2 (IL-2) thoughtto boost the immune system against cancer cells and is indicated for thetreatment of adults with metastatic renal cell carcinoma (metastaticRCC). Severe adverse events generally accompany this therapy at therecommended dosages. Thus, there is a need to identify improved methods.

The cytokine granulocyte macrophage colony stimulating factor (GM-CSF)enhances the adaptive immune system by enhancing antigen presentationand co-stimulation by dendritic cells (DC). Due to its immunestimulatory functions, GM-CSF has been used to augment host immunesystems against cancer and to boost the white blood cell count forpatients after chemotherapy. Provenge™ is a FDA-approved autologouscellular cancer immunotherapy treatment. Peripheral blood leukocytes ofa subject are harvested via leukapheresis. These enriched monocytes areincubated with prostatic acid phosphatase (PAP) conjugated togranulocyte macrophage colony stimulating factor (PAP-GM-CSF). GM-CSF isthought to direct the target antigen to receptors on DC precursors,which then present PAP on their cell surface in a context sufficient toactivate T cells for the cells that express PAP. Activated, PAPpresenting DCs are administered to the subject to elicit an immuneresponse retarding cancer growth. This strategy requires isolation andexpansion of cells of the subject, and typically treatment does notentirely clear the subject of cancer or tumors. Thus, there is a need toidentify improved methods.

Interleukin 4 (IL-4) is a γ-chain cytokine. It serves as a signal toactivate and elicit antibody class switching by B lymphocytes andconverts naïve helper T lymphocytes to active T lymphocytes and thenexpand their population. U.S. Pat. No. 6,838,081 reports enhancing thedevelopment of antigen presenting cells from precursor cells byadministering a combination of IL-4 and GM-CSF. See also U.S. PatentApplication 2004/0072299 and Hikino et al., ANTICANCER RESEARCH 24:1609-1616 (2004).

GIFT fusokines are the fused proteins derived from GM-CSF and common γchain interleukin fusion transgenes and may either suppress or enhancehost immune response. Stagg et al., Molecular Therapy, 2004, 9,S133-S133, disclose a GM-CSF/IL-2 Fusion (GIFT2). See also Penafuete etal., Cancer Res. 2009, 69(23):9020-8; Rafei et al., Nat Med. 2009,15(9):1038-45; Williams and Park, Cancer, 1991, 67(10 Suppl):2705-7; WO2005/0053579; WO 2005/026820; WO 2008/0014612; and U.S. Pat. Nos.7,323,549; 7,217,421; 6,617,135; and 5,108,910.

SUMMARY

In certain embodiments, this disclosure relates to conjugates comprisinga polypeptide of GM-CSF and a polypeptide IL-4. Typically, the GM-CSFand IL-4 are connected by a linker, e.g., polypeptide. In certainembodiments, the disclosure relates to isolated nucleic acids encodingthese polypeptide conjugates, vectors comprising nucleic acid encodingpolypeptide conjugates, and protein expression systems comprising thesevectors such as infectious viral particles and host cells comprisingsuch nucleic acids.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising conjugates and vectors disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thedisclosure relates to vaccines comprising conjugates and vectorsdisclosed herein and an antigen and optionally an adjuvant. Typically,the antigen is contained in a live attenuated virus, killed virus, avirus-like particle, virosome, cancerous cell, lipid bilayer structurewith a surface antigen, and the antigen is typically a viral protein orglycoprotein, bacteria, or bacterial antigen, or tumor associatedantigen. In certain embodiments, the antigen is conjugated to adendritic cell marker.

In certain embodiments, the disclosure contemplates a method of mixingconjugates herein with B cells under conditions such that activatednormal B cells can produce chemokine CCL3 or elevated levels ofINF-gamma. In certain embodiments, the B-cells are chronic lymphoidleukemia B-cells. In certain embodiments, the mixing is in vitro or invivo. In certain embodiments, in vitro activated B cells areadministered to a subject, e.g., from which the B cells were originallyobtained in an amount to effectively treat or prevent cancer.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral, bacterial, or parasitic infection comprisingadministering an effective amount of a pharmaceutical compositioncomprising a conjugate or vector disclosed herein optionally incombination with an antigen and optionally an adjuvant. In certainembodiments, the subject is at risk or, exhibiting symptoms of, ordiagnosed with a viral infection, such as a chronic viral infection.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection comprising administering an effectiveamount of a vaccine comprising a conjugate disclosed herein to a subjectin need thereof.

In certain embodiments, the subject is diagnosed with influenza A virusincluding subtype H1N1, influenza B virus, influenza C virus, rotavirusA, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus,human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19,molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cellpolyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocyticchoriomeningitis virus (LCMV), yellow fever virus, measles virus, mumpsvirus, respiratory syncytial virus, rinderpest virus, Californiaencephalitis virus, hantavirus, rabies virus, ebola virus, marburgvirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2),varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus(CMV), herpes lymphotropic virus, roseolovirus, or Kaposi'ssarcoma-associated herpesvirus, hepatitis A, hepatitis B, hepatitis C,hepatitis D, hepatitis E or human immunodeficiency virus (HIV).

In certain embodiments, the disclosure relates to administering aconjugate or vector disclosed herein in combination with anotherantiviral agent such as abacavir, acyclovir, acyclovir, adefovir,amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla,boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine,docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, interferon type III, interferon type II, interferon type I,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a,penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir,ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, stavudine,tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir,tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc,vidarabine, viramidine zalcitabine, zanamivir, and/or zidovudine.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering a pharmaceutical compositioncomprising a conjugate or vector disclosed herein to a subject in needthereof.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering autologous blood cellsactivated with a cancer antigen conjugated to GM-CSF in combination witha conjugate disclosed herein to a subject in need thereof.

In certain embodiments, the disclosure relates to methods of activatingperipheral blood cells comprising mixing peripheral blood cells with aconjugate disclosed herein comprising a tumor associated antigen/cancermarker under conditions such that increase expression of CD54 occurs. Incertain embodiments, the disclosure relates to product produced bymixing peripheral blood cells and with a conjugate disclosed hereinunder conditions such that increase expression of CD54 occurs. Incertain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering an effective amount of aproduct made by mixing peripheral blood cells with a conjugate disclosedherein to subject from whom the peripheral blood cells were obtained.

In certain embodiments, the disclosure relates to methods of producingcompositions comprising isolated bone marrow and/or bone marrow stemcells comprising administering conjugates and compositions disclosedherein, e.g., GIFT4, GIFT4 in combination with B cells, or isolatedcells activated with GIFT4, optionally in combination with B cells, to asubject. In certain embodiments, the disclosure relates to the treatmentor prevention of irradiation-caused bone marrow failure comprisingadministering conjugates and compositions disclosed herein to a subjectin need thereof.

In certain embodiments, the disclosure relates to methods of increasingthe production of bone marrow and/or bone marrow stem cells comprisingadministering an effective amount of a conjugate disclosed herein tosubject. In certain embodiments, the disclosure relates to a productmade by the process of isolating bone marrow and/or bone marrow stemcells from a subject administered with a conjugate disclosed herein.

In certain embodiments, the disclosure relates to methods of increasingthe production of bone marrow stem cells comprising administering aneffective amount of a conjugate disclosed herein in combination with Bcells to subject that is B cell deficient.

In certain embodiments, the subject is diagnosed with a B-cellimmunodeficiency, defects of B-cell development/immunoglobulinproduction, excessive/uncontrolled B-cell proliferation, leukemia,chronic lymphocytic leukemia, lymphoma, follicular non-hodgkin'slymphoma, diffuse large B cell lymphoma, or lupus.

In certain embodiments, the disclosure relates to a product made by theprocess of isolating bone marrow and/or bone marrow stem cells from asubject administered with a conjugate disclosed herein in combinationwith B cells.

In certain embodiments, the disclosure relates to a compositioncomprising isolated bone marrow cells and/or bone marrow stem cells anda conjugate disclosed herein, e.g., GM-CSF and IL-4 conjugate. Incertain embodiments, the bone marrow cells and/or bone marrow stem cellsare obtain from a subject that was previously administered the conjugateunder conditions such that the bone marrow cells and/or bone marrow stemcells increase proliferation.

In certain embodiments, the disclosure relates to methods of treating orpreventing bone marrow failure comprising administering an effectiveamount of a product made by mixing bone marrow cells with a conjugatedisclosed herein to subject. In certain embodiments, the bone marrowfailure is caused by irradiation. In certain embodiments, the productmade by mixing bone marrow cells with a conjugate disclosed herein isadministered in combination with a conjugate disclosed herein tosubject. In certain embodiments, the conjugate is GIFT4. In certainembodiments, the bone marrow cells were obtained from the subject.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering an effective amount of aproduct made by mixing bone marrow cells with a conjugate disclosedherein to subject. In certain embodiments, the product made by mixingbone marrow cells with a conjugate disclosed herein is administered incombination with a conjugate disclosed herein to subject. In certainembodiments, the conjugate is GIFT4. In certain embodiments, the bonemarrow cells were obtained from the subject.

In some embodiments, the disclosure relates to a method of treating orpreventing cancer comprising by administering a pharmaceuticalcomposition comprising conjugates or vector disclosed herein to asubject diagnosed with, exhibiting symptoms of, or at risk of cancerwherein the cancer is a hematological malignancy such as a leukemia orlymphoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia(AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma(SLL), chronic myelogenous leukemia, acute monocytic leukemia (AMOL),Hodgkin's lymphomas, and non-Hodgkin's lymphomas such as Burkittlymphoma, B-cell lymphoma and multiple myeloma. Other contemplatedcancers include cervical, ovarian, colon, breast, gastric, lung, skin,ovarian, pancreatic, prostate, head, neck, and renal cancer.

Within any of the cancer management methods disclosed herein, theconjugate or vector may be administered in combination with ananti-cancer agent such as gefitinib, erlotinib, docetaxel, cis-platin,5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate,cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin,daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin andmithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol,taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin,bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, fulvestrant, bicalutamide, flutamide, nilutamide,cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole,letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab,cetuximab, dasatinib, imatinib, bevacizumab, combretastatin,thalidomide, and/or lenalidomide or combinations thereof. In certainembodiments, combination therapies with proleukin (recombinant humanIL-2) and or interferon alpha are contemplated.

In certain embodiments, the disclosure relates to gene therapiescomprising administering vectors comprising nucleic acid encodingconjugates disclosed herein to a subject in need thereof. In certainembodiments, the nucleic acids are isolated and/or purified from theirnatural state or translated to a non-naturally occurring form such ascDNA.

In certain embodiments, the disclosure contemplates incorporatingconjugates disclosed herein into the surfaces of particles, e.g., cells,liposomes, micelles, vesicles, bilayer structures, virosomes, andvirus-like particles. The conjugates may be linked to lipophilicmoieties, e.g., fatty acids and GPI. In one example, the disclosurecontemplates a GPI anchored conjugate comprising GPI, GM-CSF, IL-4, andoptionally an antigen, adjuvant, or other polypeptide. It iscontemplated that these particles may contain other surfacepolypeptides, antigens and co-stimulatory molecules such as B7-1, B7-2,ICAM-1, and/or IL-2. It is contemplated that these particles may be usedin all the applications conjugates disclosed herein are mentioned.

Within certain embodiments, any of the conjugates disclosed herein maybe further conjugated to an adjuvant, cytokine, co-stimulatory molecule,antigen, protein, or glycoprotein. In certain embodiments, the antigenis a viral protein or a cancer marker.

In certain embodiments, the cancer marker is selected from PAP(prostatic acid phosphatase), prostate-specific antigen (PSA), (PSMA)prostate-specific membrane antigen, early prostate cancer antigen-2(EPCA-2), AKAP-4 (A kinase [PRKA] anchor protein 4), NGEP (new geneexpressed in prostate), PSCA (prostate stem cell antigen), STEAP(six-transmembrane epithelial antigen of the prostate), MUC 1 (mucin 1),HER-2, BCL-2, MAGE antigens such as CT7, MAGE-A3 and MAGE-A4, ERKS,G-protein coupled estrogen receptor 1, CA15-3, CA19-9, CA 72-4, CA-125,carcinoembryonic antigen, CD20, CD31, CD34, PTPRC (CD45), CD99, CD117,melanoma-associated antigen (TA-90), peripheral myelin protein 22(PMP22), epithelial membrane proteins (EMP-1, -2, and -3), HMB-45antigen, MART-1 (Melan-A), S100A1, and S100B or fragments or mutatedforms thereof.

In certain embodiments, the viral antigen is selected from an influenzavirus hemagglutinin and neuraminidase; cytomegalovirus glycoprotein gB,p28, p38, p50, p52, p65, and p150; Borrelia p41; HIV nef, integrase,gag, protease, tat, env, p31, p17, p24, p31, p55, p66, gp32, gp36, gp39,gp41, gp120, and gp160; SIV p55; HBV core, surface antigen, andaustralian antigen; HCV core nucleocapsid, NS3, NS4, and NS5; Dengue envand NS1; EBV early antigen, p18, p23, gp125, nuclear antigen (EBNA)-1,EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), latentmembrane proteins (LMP)-1, LMP-2A and LMP-2B; and herpes simplex virusgD and gG or fragments or mutated forms thereof.

In certain embodiments, the adjuvant or cytokine is selected from IL-2,IL-12, IL-15, IL-7, IL-18, IL-21, IL-27, IL-31, IFN-alpha, flagellin,unmethylated, CpG oligonucleotide, lipopolysaccharides, lipid A, andheat stable antigen (HSA).

In certain embodiments, the disclosure contemplates administration ofpharmaceutical products comprising conjugates disclosed herein byintravenous (IV), subcutaneous (SC), or intraperitoneal (IP)administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the GIFT4 protein (SEQ ID NO: 7). Amino acidsequence of murine GIFT4 protein-GM-CSF amino acids (SEQ ID NO:1), thelinker S, and IL-4 amino acids (SEQ ID NO: 2).

FIG. 1B shows the predicted three-dimensional structure of GIFT4protein.

FIG. 1C shows intact GIFT4 protein (50 KDa) expressed by 293T cells,detected by Western blot with both anti-mouse GM-CSF and IL-4antibodies.

FIG. 1D shows data when GM-CSF-responsive JAWSII cells were treated withGIFT4 or recombinant GM-CSF or IL-4 for 72 hours, medium served ascontrol. Cell growth was analyzed by MTT assay.

FIG. 1E shows data when IL-4-responsive CT.h4S cells were treated.

FIG. 2A shows data on the phenotype of GIFT4-treated B cells. Inductionof B cell proliferation by GIFT4 stimulation. The cells were culturedfor 4 days. Combined use of recombinant GM-CSF and IL-4 served ascontrol.

FIG. 2B shows data where cells were labeled with CFSE dye. Cellsdivision cycles were presented as individual peaks.

FIG. 2C shows sata on surface markers of GIFT4-treated B cells (blackcolor filled) compared to untreated B cells (gray color filled) orantibody isotype control (black line).

FIG. 2D shows data indicating down-regulation of co-stimulatorymolecules CD80 and CD86, and immunoglobulin switch from IgM to IgG in Bcells after BCR cross-linking with anti-mouse IgM antibodies in presenceof GIFT4 (black color filled). Combined recombinant GM-CSF and IL-4(gray color filled) served as control. Black line only is the antibodyisotype control.

FIG. 3A shows data on the secretome of GIFT4-activated B cells.Phosphorylation of STAT1, STAT3, STAT5 and STAT6 activated by GIFT4stimulation (20 minutes), detected by Western blot.

FIG. 3B shows data on the secretion of cytokines and chemokines byGIFT4-treated B cells; cytokine concentration was analyzed by luminexassay.

FIG. 3C shows data on the secretion of cytokines and chemokines byGIFT4-treated B cells; cytokine concentration was analyzed by luminexassay.

FIG. 3D shows induction of GM-CSF+ innate response activator (IRA, #42)B-cells profiled by FACS.

FIG. 3E shows calculated percentage of GM-CSF-producing B cells. Datawere represented from three independent experiments.

FIG. 4A shows data on the suppression of melanoma tumor growth in vivoby GIFT4. Splenomegaly in C57BL/6J mice treated with GIFT4.Administration of recombinant GM-CSF and IL-4 served as control.

FIG. 4B shows data when splenocytes per spleen were isolated andcounted. B cells and T cells were profiled by FACS analysis. Totalsplenic B cells or T cells were calculated.

FIG. 4C shows data when B16F0 melanoma cells were subcutaneouslyimplanted into C57BL/6J mice. On day 5, the mice were treated with GIFT4protein by intravenous injection; mice administrated with GM-CSF plusIL-4 or untreated mice served as control.

FIG. 4D shows data when GIFT4-expressing B 16F0 melanoma cells weresubcutaneously implanted in B6 mice. Mixture of B16F0-GMCSF plusB16F0-IL-4 melanoma cells or wild type B16F0 cells served as controlcells. Tumor size was measured. Five mice are in each group oftreatment; data are represented from three independent experiments.

FIG. 5A shows data on the B-cell dependent tumorcidal activity elicitedby GIFT4. B16F0-GIFT4 melanoma cells were subcutaneously implanted intoRag1 knockout, B-cell deficient (μMT), or wild type B6 mice. Rapidgrowth of melanoma tumors observed in absence of adaptive immunity.

FIG. 5B shows data for CD4 or CD8 T cell-deficient mice.

FIG. 5C shows data when T cells were co-cultured with B cells stimulatedwith GIFT4, individual or combined cytokines. IFN-γ production in theculture supernatant was measured with ELISA kit.

FIG. 5D shows data when B16F0-GIFT4 tumor cells were subcutaneouslyinjected into IFN-γ−/−, IL-10−/−, IL-12−/− or wild type mice. Five miceare in each group; data are represented from three independentexperiments.

FIG. 6A shows data on the antigen-melanoma specific antibody productionenhanced by GIFT4. Time schedule of OVA administration in C57BL/6J mice.OVA were injected into mice supplemented with GIFT4 protein or combinedrecombinant GM-CSF and IL-4. Mice without cytokine treatment served asblank control.

FIG. 6B shows data when spleens were harvested from the mice, and Bcells were purified from splenocytes. OVA-specific IgG-secreting cellsper 50,000 B cells were determined by ELISpot assay.

FIG. 6C shows data when C57BL/6J mice (gray color filled) or B-celldeficient mice (unfilled gray line) were immunized with B16F0-GIFT4cells. PBS-treated mice served as controls (unfilled black line). Serafrom the mice were used as primary antibody for FACS analysis with B16F0melanoma cells followed by incubation of PE-conjugated anti-mouse IgGsecondary antibodies.

FIG. 6D shows data for mean fluorescent intensity of B 16F0 melanomacells treated with serum from mice in each group. Five mice are in eachgroup; data are represented from three independent experiments.

FIG. 6E shows data when FcγR−/−, B-cell deficient μMT or wild typeC57BL/6J mice were implanted with B16F0-GIFT4 melanoma cells. Tumorgrowth was monitored and measured. Five mice are in each group; data arerepresented from three independent experiments.

FIG. 7A shows data on the inhibition of melanoma tumor growth byadoptive-transferred B cells. Immunized mice or unimmunized controlC57BL/6J mice were challenged with B16F0 melanoma cells on day 30, andtumor growth was monitored and measured.

FIG. 7B shows data when μMT mice implanted with B16F0-GIFT4 tumor cellswere adaptively transferred with B cells isolated from immunizedC57BL/6J mice. Mice without B cell transfer served as control. Five miceare in each group of treatment; data are represented from threeindependent experiments.

FIG. 8A shows data on the induction of splenic cell proliferation byGIFT4 stimulation. Splenocytes isolated from C57BL/6J mice were treatedwith GIFT4 or combined recombinant GM-CSF and IL-4 for 5 days. Expansionof splenocytes aggregated as clusters.

FIG. 8B shows data when cells were collected and subjected to FACS withanti-mouse B220 and anti-CD3 antibodies, typical antibodies for murine Bcells and T cells.

FIG. 9A shows data on GIFT4-triggered expansion of GM-CSF⁺ IRA-likecells in vivo. Purified splenic B cells from mice treated with GIFT4 orcombined use of GM-CSF and IL-4 were subject to intracellular stainingof GM-CSF, followed by FACS.

FIG. 9B shows the calculated percentage of GM-CSF-producing B cells wascalculated. Data were represented from two independent experiments.

FIG. 10 illustrates a proposed model of GIFT4 immune functions on CLLcells. GIFT4 has potent anti-CLL immune function by reprogramingleukemic B cells into anti-CLL effectors and helpers, which drive theexpansion of IFN-γ, NK, and T cells, as well as NKT cells.

FIG. 11A illustrates human GIFT4 protein (SEQ ID NO: 8), a polypeptideof GM-CSF, MWLQSLLLLGTV ACSISAPARS PSPSTQPWEHVNAI QEARRLLN LSRDTAAEMNETVEVISE MFDLQEPTC LQTRLELYKQGL RGSLTKLKGPLTMMASH YKQHCPPTPETSCATQTITFESF KENLKDFLL VIPFDCWEPVQE (SEQ ID NO: 6), an S linker and, humanisoform 1 of IL-4 (SEQ ID NO: 3).

FIG. 11B shows a 3D structure of human GIFT4 protein.

FIG. 11C shows data when GIFT4 protein expressed by genetic-modified293T-GIFT4 cells was detected by Western blot.

FIG. 11D shows GIFT4 has strong biological signaling activities of IL-4signaling and induces proliferation of IL-4-responder CT.h4S cells.

FIG. 12A shows data on the activation of CLL-B cells by human GIFT4protein. Purified CLL B cells were stimulated with human GIFT4 proteinor combined GM-CSF plus IL-4.

FIG. 12B shows data indicating GIFT4 triggers hyper-phosphorylation ofSTATS in CLL B cells, but not STAT1, 3 and 6.

FIG. 13A shows data on GIFT4-converted CLL B cells have unique surfacemarkers and secretome. GIFT4 reprograms leukemic B cells intoantigen-presenting cells, which are CD5⁺, CD19⁺, CD23⁺, CD40⁺, CD54⁺,CD80⁺, CD86⁺, MHC I/II⁺, but CD124^(low).

FIG. 13B shows data indicating GIFT4-treated CLL B cells secrete IL-1β,IL-6, ICAM1 and massive amounts of IL-2.

FIG. 14A shows data indicating GIFT4-reprogramed CLL B cells primeautologous NK and T cell immune response. CD5⁺CD19⁺ B cells are themajor component of CLL cells, CD3⁺ T cells and CD16⁺ NK cells are theminor populations (upper panels). GIFT4 treatment robustly propelled theexpansion of NK and T cells, as well as CD3⁺CD16⁺ NKT cells (middlepanels), which produce massive IFN-γ (low panels).

FIG. 14B shows data for IFN-γ.

FIG. 14C shows data when co-culture of GIFT4-treated CLL cells withprimary autologous CLL cells from patients led to the killing of primarypCLL cells in vitro.

FIG. 15A shows data indicating GIFT4 treatment increases bone marrowstem cells (BMSC) in B6 mice (Lin⁻SCA-1⁺CD117⁺). GIFT4 (20 ng/day) orcontrol cytokines were injected into B6 Mice for 6 Days. Bone marrowcells were isolated and subject to FACS analysis with lineage markersand stem cell markers. The number of BMSC per femur was calculated.Experiments were repeated three, 5 mice each group.

FIG. 15B shows data on the surface markers that are Lin⁻Sca-1⁺Ckit⁺.

FIG. 16 shows data indicating B-cell deficiency abrogates the increaseof BMSC in μMT mice. GIFT4 (20 ng/day) or control cytokines was injectedinto μMT B cell-deficient mice for 6 Days. Bone marrow cells wereisolated and subject to FACS analysis with lineage markers and stem cellmarkers (See FIG. 15). The number of BMSC per femur was calculated.Experiments were repeated twice, 5 mice each group.

FIG. 17 shows data indicating adoptive transfer of B cells promotes BMSCexpansion in μMT mice after GIFT4 treatment. Adoptive transfer of Bcells (2×10⁷ cells/mouse) into μMT B cell-deficient mice combined withGIFT4 treatment for 6 days. Bone marrow cells were isolated and subjectto FACS analysis with lineage markers and stem cell markers (See FIG.15). The number of BMSC per femur was calculated. Experiments wererepeated twice, 5 mice each group.

FIG. 18 shows data indicating GIFT4 programmed BMSC have similarfunctions with the BMSC from naïve mice. B6 mice were irradiated at 11Gy (5.5+5.5 Gy, 3 hr interval), then injected i.v. with BMSC purifiedfrom mGIFT4-treated or naïve mice. Mice weight loss and survival weremonitored. Experiments were repeated twice, 5 mice each group.

FIG. 19 shows data indicating delivery of GIFT4 plus B cells amelioratesirradiation-caused bone marrow failure. B6 mice were irradiated at 11 Gy(5.5+5.5 Gy, 3 hr interval), then injected i.v. with mGIFT4 (20ng/mouse/day for 6 days), or plus B cells (2×106 cells/mouse). Micewithout treatment served as controls. Mice weight loss and survival weremonitored. Experiments were repeated twice, 5 mice each group.

DETAILED DESCRIPTION

Common γ chain interleukin cytokines include IL-2, IL-4, IL-7, IL-9,IL-15 and IL-21, and have important roles in the activation anddifferentiation of lymphocytes. IL-2 has strong immune modulatoryproperties on T cells, and had been approved by FDA as the firstinterleukin immunotherapeutic agent to use to treat patients withadvanced kidney cancer and metastatic melanoma. Unfortunately, IL-2immunotherapy even combined with various chemotherapy drugs has notshown significant improvement of survival time in cancer patients. Inaddition, IL-2 has frequent, often serious and sometimes fatal sideeffects due to capillary leakage.

GIFT2 shows anti-tumor activities via induction of tumor-killer NKcells. GIFT2 affects tumoricidal dendritic cells. In contrast, GIFT15(derived from GM-CSF and IL-15) has immune suppressive function toabrogate inflammatory response in multiple sclerosis. Here, a fusokinederive from GM-CSF and IL-4 (GIFT4) is show to be a cancerimmunotherapeutic agent. Functionally different from GM-CSF and IL-4that induces monocytes to differentiate into dendritic cells in vitro,GIFT4 directly elicits adaptive B-cell immune response and consequent Tcell immunity.

GM-CSF-IL-4 Fusokine (GIFT4)

FIG. 1 provides for an embodiment of this disclosure comprising a GM-CSFsequence and a murine IL-4 sequence and FIG. 11 provides s humansequence. In certain embodiments, the disclosure contemplates a fusokinewith a recombinant human form such as isoform 1 which is amino acidssequence MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTELTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRLDRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS (SEQ ID NO: 3) orisoform 2 which is amino acid sequence MGLTSQLLPP LFFLLACAGN FVHGHKCDITLQEIIKTLNS LTEQKNTTEK ETFCRAATVL RQFYSHHEKD TRCLGATAQQ FHRHKQLIRFLKRLDRNLWG LAGLNSCPVK EANQSTLENFLERLKTIMRE KYSKCSS (SEQ ID NO: 4).Isoform 2 lacks an in-frame exon in the 5′ region, compared to variant1, resulting an isoform (2) that lacks an internal region, as comparedto isoform 1.

The present disclosure encompasses fusion proteins involving full-lengthpre-processed forms, as well as mature processed forms, fragmentsthereof and variants of each or both of the GM-CSF and IL-4 entitieswith linker amino acids, including allelic as well as non-naturallyoccurring variants. In addition to naturally-occurring allelic variantsof the GM-CSF and IL-4 entities that may exist in the population, theskilled artisan will further appreciate that changes (i.e. one or moredeletions, additions and/or substitutions of one or more amino acid) canbe introduced by mutation using classic or recombinant techniques toeffect random or targeted mutagenesis. A suitable variant in use in thepresent disclosure typically has an amino acid sequence having a highdegree of homology with the amino acid sequence of the correspondingnative cytokine. In one embodiment, the amino acid sequence of thevariant cytokine in use in the fusion protein of the disclosure is atleast 70%, at least about 75%, at least about 80%, at least about 90%,typically at least about 95%, more typically at least about 97% and evenmore typically at least about 99% identical to the corresponding nativesequence, e.g., SEQ ID NO: 3 or 4. In certain embodiments, such nativesequence is of human GM-CSF and/or human IL-4.

Percent identities between amino acid or nucleic acid sequences can bedetermined using standard methods known to those of skill in the art.For instance for determining the percentage of homology between twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes. The amino acid residues at corresponding amino acid positionsare then compared. Gaps can be introduced in one or both amino acidsequence(s) for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes. When a position in the firstsequence is occupied by the same amino acid residue as the correspondingposition in the second sequence, then the sequences are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps which need to be introduced foroptimal alignment and the length of each gap. The comparison ofsequences and determination of percent identity and similarity betweentwo sequences can be accomplished using a mathematical algorithm (e.g.Computational Molecular Biology, 1988, Ed Lesk A M, Oxford UniversityPress, New York; Biocomputing: Informatics and Genome Projects, 1993, EdSmith D. W., Academic Press, New York; Computer Analysis of SequenceData, 1994, Eds Griffin A. M. and Griffin H. G., Human Press, NewJersey; Sequence Analysis Primer, 1991, Eds Griskov M. and Devereux J.,Stockton Press, New York). Moreover, various computer programs areavailable to determine percentage identities between amino acidsequences and between nucleic acid sequences, such as GCG™ program(available from Genetics Computer Group, Madison, Wis.), DNAsis™ program(available from Hitachi Software, San Bruno, Calif.) or the MacVector™program (available from the Eastman Kodak Company, New Haven, Conn.).

Suitable variants of GM-CSF and IL-4 entities for use in the presentdisclosure are biologically active and retain at least one of theactivities described herein in connection with the correspondingpolypeptide. Typically, the therapeutic effect (e.g. anti-tumoractivity, by-pass of tumor-induced immune energy) is preserved, althougha given function of the polypeptide(s) may be positively or negativelyaffected to some degree, e.g. with variants exhibiting reducedcytotoxicity or enhanced biological activity. Amino acids that areessential for a given function can be identified by methods known in theart, such as by site-directed mutagenesis. Amino acids that are criticalfor binding a partner/substrate (e.g. a receptor) can also be determinedby structural analysis such as crystallization, nuclear magneticresonance and/or photoaffinity labeling. The resulting variant can betested for biological activity in assays such as those described above.

For example, in one class of functional variants, one or more amino acidresidues are conservatively substituted. A “conservative amino acidsubstitution” is one in which the amino acid residue in the nativepolypeptide is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. Typically, substitutions are regarded asconservative when the replacement, one for another, is among thealiphatic amino acids Ala, Val, Leu, and Ile; the hydroxyl residues Serand Thr; the acidic residues Asp and Glu; the amide residues Asn andGln; the basic residues Lys and Arg; or the aromatic residues Phe andTyr. Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a cytokine coding sequence, such as bysaturation mutagenesis, and the resultant mutant can be screened for itsbiological activity as described herein to identify mutants that retainat least therapeutic activity.

Although the GM-CSF and IL-4 entities can be directly fused in thefusion protein of the disclosure, it is however typical to use a linkerfor joining GM-CSF and IL-4. The purpose of the linker is to allow thecorrect formation, folding and/or functioning of each of the GM-CSF andIL-4 entities. It should be sufficiently flexible and sufficiently longto achieve that purpose. Typically, the coding sequence of the linkermay be chosen such that it encourages translational pausing andtherefore independent folding of the GM-CSF and IL-4 entities. A personskilled in the art will be able to design suitable linkers in accordancewith the disclosure. The present disclosure is, however, not limited bythe form, size or number of linker sequences employed. Multiple copiesof the linker sequence of choice may be inserted between GM-CSF andIL-4. The only requirement for the linker sequence is that itfunctionally does not adversely interfere with the folding and/orfunctioning of the individual entities of the fusion protein. Forexample, a suitable linker is 1 to 5 or 5 to 50 amino acid long and maycomprise amino acids such as glycine, serine, threonine, asparagine,alanine and proline (see for example Wiederrecht et al., 1988, Cell 54,841; Dekker et al., 1993, Nature 362, 852; Sturm et al., 1988, Genes andDev. 2, 1582; Aumailly et al., 1990 FEBS Lett. 262, 82). Repeatscomprising serine and glycine residues are typical in the context of thedisclosure. Specific examples of suitable linkers consists of two orthree or more (e.g. up to eight or more) copies of the sequenceGly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 5). It will be evident that thedisclosure is not limited to the use of these particular linkers.

The disclosure further includes fusion proteins which comprise, oralternatively consist essentially of, or alternatively consist of anamino acid sequence which is at least 70%, 75%, 80%, 90%, 95%, 97%, 99%homologous or even better 100% homologous (identical) to all or part ofany of the amino acid sequences recited in SEQ ID NO: 1-6.

In the context of the present disclosure, a protein “consists of” anamino acid sequence when the protein does not contain any amino acidsbut the recited amino acid sequence. A protein “consists essentially of”an amino acid sequence when such an amino acid sequence is presenttogether with only a few additional amino acid residues, typically fromabout 1 to about 50 or so additional residues. A protein “comprises” anamino acid sequence when the amino acid sequence is at least part of thefinal (i.e. mature) amino acid sequence of the protein. Such a proteincan have a few up to several hundred additional amino acids residues.Such additional amino acid residues can be naturally associated witheach or both entities contained in the fusion or heterologous aminoacid/peptide sequences (heterologous with respect to the respectiveentities). Such additional amino acid residues may play a role inprocessing of the fusion protein from a precursor to a mature form, mayfacilitate protein trafficking, prolong or shorten protein half-life orfacilitate manipulation of the fusion protein for assay or production,among other things. Typically, the fusion proteins of the disclosurecomprise a signal peptide at the NH₂-terminus in order to promotesecretion in the host cell or organism. For example, the endogenoussignal peptide (i.e. naturally present in the cytokine present at theNH₂ terminus of said fusion) can be used or alternatively a suitableheterologous (with respect to the cytokine in question) signal peptidesequence can be added to the cytokine entity present at the NH₂ terminusof the fusion or inserted in replacement of the endogenous one.

In the context of the disclosure, the fusion proteins of the disclosurecan comprise cytokine entities of any origin, i.e. any human or animalsource (including canine, avian, bovine, murine, ovine, feline, porcine,etc). Although “chimeric” fusion proteins are also encompassed by thedisclosure (e.g. one cytokine entity of human origin and the other of ananimal source), it is typical that each entity be of the same origin(e.g. both from humans).

The fusion proteins of the present disclosure can be produced bystandard techniques. Polypeptide and DNA sequences for each of thecytokines involved in the fusion protein of the present disclosure arepublished in the art, as are methods for obtaining expression thereofthrough recombinant or chemical synthetic techniques. In anotherembodiment, a fusion-encoding DNA sequence can be synthesized byconventional techniques including automated DNA synthesizers. Then, theDNA sequence encoding the fusion protein may be constructed in a vectorand operably linked to a regulatory region capable of controllingexpression of the fusion protein in a host cell or organism. Techniquesfor cloning DNA sequences for instance in viral vectors or plasmids areknown to those of skill in the art (Sambrook et al, 2001, “MolecularCloning. A Laboratory Manual”, Laboratory Press, Cold Spring HarborN.Y.). The fusion protein of the disclosure can be purified from cellsthat have been transformed to express it.

The present disclosure also provides a nucleic acid molecule encodingthe fusion protein of the disclosure. Within the context of the presentdisclosure, the term “nucleic acid” and “polynucleotide” are usedinterchangeably and define a polymer of nucleotides of any length,either deoxyribonucleotide (DNA) molecules (e.g., cDNA or genomic DNA)and ribonucleotide (RNA) molecules (e.g., mRNA) and analogs of the DNAor RNA generated using nucleotide analogs (see U.S. Pat. No. 5,525,711and U.S. Pat. No. 4,711,955 as examples of nucleotide analogs). Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides mayalso be interrupted by non-nucleotide elements. The nucleic acidmolecule may be further modified after polymerization, such as byconjugation with a labeling component. The nucleic acid, especially DNA,can be double-stranded or single-stranded, but typically isdouble-stranded DNA. Single-stranded nucleic acids can be the codingstrand (sense strand) or the non-coding strand (anti-sense strand).

The nucleic acid molecules of the disclosure include, but are notlimited to, the sequence encoding the fusion protein alone, but maycomprise additional non-coding sequences, for example introns andnon-coding 5′ and 3′ sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and mRNA stability. For example, the nucleic acid molecule ofthe disclosure can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank (i.e.sequences located at the 5′ and 3′ ends) or are present within thegenomic DNA encoding GM-CSF and IL-4 entities.

According to a typical embodiment, the present disclosure providesnucleic acid molecules which comprise, or alternatively consistessentially of, or alternatively consist of a nucleotide sequenceencoding all or part of an amino acid sequence encoding a fusion proteinwhich is at least about 70%, at least about 75%, at least about 80%, atleast about 90%, at least about 95%, typically at least about 97%, moretypically at least about 99% homologous or even more typically 100%homologous to any of the amino acid sequences shown in SEQ ID NO: 1-6.

In another embodiment, a nucleic acid molecule of the disclosurecomprises a nucleic acid molecule which is a complement of all or partof a nucleotide sequence encoding the fusion protein shown in any of SEQID NO: 1-6. A nucleic acid molecule which is complementary to thenucleotide sequence of the present disclosure is one which issufficiently complementary such that it can hybridize to thefusion-encoding nucleotide sequence under stringent conditions, therebyforming a stable duplex. Such stringent conditions are known to thoseskilled in the art. A typical, non-limiting example of stringenthybridization conditions are hybridization in 6 times sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2 times SSC, 0.1% SDS at 50-65 C. In one embodiment, thedisclosure pertains to antisense nucleic acid to the nucleic acidmolecules of the disclosure. The antisense nucleic acid can becomplementary to an entire coding strand, or to only a portion thereof.

In still another embodiment, the disclosure encompasses variants of theabove-described nucleic acid molecules of the disclosure e.g., thatencode variants of the fusion proteins that are described above. Thevariation(s) encompassed by the present disclosure can be created byintroducing one or more nucleotide substitutions, additions and/ordeletions into the nucleotide sequence by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Followingmutagenesis, the variant nucleic acid molecule can be expressedrecombinantly as described herein and the activity of the resultingprotein can be determined using, for example, assays described herein.Alternatively, the nucleic acid molecule of the disclosure can bealtered to provide preferential codon usage for a specific host cell(for example E. coli; Wada et al., 1992, Nucleic Acids Res. 20,2111-2118). The disclosure further encompasses nucleic acid moleculesthat differ due to the degeneracy of the genetic code and thus encodefor example the same fusion protein as any of those shown in SEQ ID NO:1-6.

Another embodiment of the disclosure pertains to fragments of thenucleic acid molecule of the disclosure, e.g. restriction endonucleaseand PCR-generated fragments. Such fragments can be used as probes,primers or fragments encoding an immunogenic portion of the fusionprotein.

The nucleic acid molecules of the present disclosure can be generatedusing the sequence information provided herein. The nucleic acidencoding each of the GM-CSF and IL-4 entities can be cloned or amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateprobes or oligonucleotide primers according to standard molecularbiology techniques (e.g., as described in Sambrook, et al. “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001) or standard PCR amplification techniquesbased on sequence data accessible in the art (such as those providedabove in connection with the fusion proteins of the disclosure or thoseprovided in the Examples part). Fusing of the GM-CSF sequence to theIL-4 sequence may be accomplished as described in the Experimental belowor by conventional techniques. For example, the GM-CSF and IL-4 encodingsequences can be ligated together in-frame either directly or through asequence encoding a peptide linker. The GM-CSF-encoding sequence canalso be inserted directly into a vector which contains the IL-4-encodingsequence, or vice versa. Alternatively, PCR amplification of the GM-CSFand IL-4-encoding sequences can be carried out using primers which giverise to complementary overhangs which can subsequently be annealed andre-amplified to generate a fusion gene sequence.

GM-CSF and IL-4 Fusion Cytokine Triggers Conversion of B-Cells toTumoricidal Effectors

Studies herein indicate that the GIFT4 fusokine has the powerfulcapability to convert naive B-cells to tumoricidal effectors. Given itspotential impact on host B cell immune functions, fusokine GIFT4 offersa strategy for immunotherapy against a wide variety of cancers, e.g.,using a B cell-based cancer immunotherapy.

In certain embodiments the disclosure contemplates the fusokine GIFT4(derive from GM-CSF and IL-4) use as a cancer immunotherapeutic agent.Functionally different from GM-CSF and IL-4 that induces monocytes todifferentiate into dendritic cells in vitro, GIFT4 directly elicitsadaptive B-cell immune response and consequent T cell immunity. SeeGluckman et al., Cytokines, cellular & molecular therapy, 1997,3:187-196. GIFT4 fusokine is a potent activator to trigger robust B cellimmunity against tumor. Uniquely, GIFT4-B cells secrete an array ofinnate cytokines such as IL-1β, IL-6, IL-12, VEGF, GM-CSF and chemokineCCL3, but not IL-10 and IFN-γ. The pro-Th1 cytokines IL-12, IL-1β andIL-6 can promote IFN-γ production by T cells and enhance host anti-tumorimmunity. As a parental molecule of GIFT4, IL-4 stimulation only inducesstrong phosphorylation of STST6 in B cells, with little or weakphosphorylation of other STATs. However, GIFT4 protein showsgain-of-function to trigger hyper-phosphorylation of STAT1, STAT3, STAT5and STAT6 upon ligation with B cells, the former two STATs are the majordownstream signaling pathways for other IL-2 common γ chain familymembers including IL-2, IL-7, IL-9, IL-15 and IL-21. Phosphorylation ofSTAT3 is related to the production of IL-1β and IL-6. There is no reportthat STAT phosphorylation results in CCL3 secretion by normal B cells;however, BCR stimulation activates leukemic B cells to produce chemokineCCL3. See Miyauchi et al., Cancer science, 2011, 102:1236-1241. AlthoughGIFT4 protein possesses the functional activities of both GM-CSF andIL-4 components, how phosphorylation of STATs leads to the secretion ofGM-CSF and CCL3 by GIFT4-B cells remains unclear.

GIFT4-B cells have a unique phenotype expressing surface markers B220,CD19, CD40, CD80, CD86, and IgM, but not CD23. GIFT4-B cells secretehigh amount of GM-CSF. Unlike IRA-B cells that secrete high amounts ofGM-CSF (See Rauch et al., Science, 2012, 335:597-601), GIFT4-B cells areCD80⁺CD86⁺ that are typical markers for antigen-presenting cells. Thephenotype of GIFT4-B cells is also different from B1 cells and B2 cells,of which B1 cells are B220^(low), and B2 cells are CD23⁺. Unlike IRA Bcells, GIFT4-B cells also secrete IL-1β, IL-5, IL-6, IL-12, VEFG andmassive CCL3, but not IL-3 that is uniquely produced by IRA B cells. Toour knowledge, it is the first report that activated normal B cells canproduce chemokine CCL3. Therefore, GIFT4-B cells have distinctcytokine-secreting profile that can enhance IFN-γ-mediated T cellantitumor immunity, but sharing some common surface markers with IRA-Bcells. Strikingly, GIFT4-B cells have the plasticity to isotype switchfrom IgM to IgG upon BCR cross-linking; similar to IL-4 and anti-mouseIgM-treated B cells. Thus, GIFT4-B cells could function as both innateeffectors and adaptive responders against a variety of cancers, andpossible infectious pathogens.

GIFT4 fusokine triggers previously un-described B cell-dependentanti-tumor immunity in a murine model of melanoma. B cells compriseheterogeneous subpopulations of B-lymphocytes with two sides of immuneactivities either enhancing or suppressing host immunity via B cellcytokine secretion, antibody production, and cellular interaction. Earlyinvestigation showed that B cells suppress host immune response againsttumors by inhibition of T cell tumorcidial activities via IL-10 mediatedpathway. However, new emerging evidences suggest that B cells playimportant protective roles in anti-tumor immunity. Studies disclosedherein demonstrated that GIFT4 induce B cell-initiated anti-melanomaimmune response.

Administration of GIFT4 protein in vivo results in the expansion of aglobal of B cells including GM-CSF-secreting IRA-like B cells withoutexpansion IL-10-producing regulatory B cells, since GIFT4-B cells do notsecretes IL-10. GM-CSF has been shown to increase the proliferation ofantigen-activated cytotoxic T cells; chemokine CCL3 is important forrecruiting CCR5⁺ T cells in anti-tumor immunity, suggesting that GM-CSFand CCL3-producing GIFT4-B cells have potent efficacy to promote T cellimmunity against tumors. In addition, VEGF-producing B cells facilitatedendritic cells migration in vivo. Co-culture of GIFT4-B cells with Tcells markedly increase IFN-γ production by T cells further verifiesthat GIFT4-B cells actively participate in cellular immunity byaugmenting the magnitude of IFN-γ⁺ T cell response. The robust growth ofB16F0 melanoma tumors in B-cell deficient mice that possess normal Tcell function confirms that GIFT4-triggered anti-tumor immunity is Bcell-dependent. The fact that B cells can act as real antigen-presentingcells to directly license cytotoxic T cells and establish long-lastingantitumor immunity strongly indicates that GIFT4-B cells function likedendritic cells to elicit T-cell immunity against tumors.

Antigen-specific antibody production is an important protective arm of Beffector cells against infectious pathogens and cancers. IL-4 has beenshown to participate in generation and expansion of memory B cells uponantigen-BCR ligation; however IL-4 stimulation could not increase thenumber of plasma cells. See Choe et al., Journal of immunology, 1997,159:3757-3766. TLR ligands CpG oligodeoxynucleotides and LPS canactivate memory B cells and differentiate into plasma cells in vitro,but lose the capability in vivo. Dramatic expansion of antigen-specificplasma cells and augmentation of antigen-specific antibody production invivo by GIFT4 stimulation indicates that GIFT4 protein is a potentstimulator for B cell anti-tumor humoral response. In melanoma mousemodel, immunization of GIFT4-secreting B16F0 cells leads totumor-specific antibody secretion in immunized mice suggests that GIFT4is a powerful adjuvant for induction of anti-melanoma specificantibodies. The complete inhibition of melanoma tumor growth inB16F0-GIFT4 cell-immunized B6 mice indicates that GIFT4 elicits acquiredanti-tumor B cell immunity. Fc-receptor substantially contributes to theaction of cytotoxic antibodies against tumors through antibody-dependentcell-mediated cytotoxicity pathway (ADCC). Data that mice lack of Fcγreceptor could not prevent GIFT4-secreting B16F0 tumor growth furtherhighlights the participation of ADCC in GIFT4-triggered B-cellanti-melanoma immunity. In a pre-established melanoma model, adoptivetransfer of tumor-primed B cells remarkably inhibited melanoma growth inB cell deficient mice further confirms the anti-melanoma effect of GIFT4is dependent on the adoptive B-cell activity.

Pharmaceutical Compositions

As used herein the language “pharmaceutically acceptable excipient” isintended to include any and all carriers, solvents, diluents,excipients, adjuvants, dispersion media, coatings, antibacterial andantifungal agents, and absorption delaying agents, and the like,compatible with pharmaceutical administration.

Suitably, the pharmaceutical composition of the disclosure comprises acarrier and/or diluent appropriate for its delivering by injection to ahuman or animal organism. Such carrier and/or diluent is non-toxic atthe dosage and concentration employed. It is selected from those usuallyemployed to formulate compositions for parental administration in eitherunit dosage or multi-dose form or for direct infusion by continuous orperiodic infusion. It is typically isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as provided bysugars, polyalcohols and isotonic saline solutions. Representativeexamples include sterile water, physiological saline (e.g. sodiumchloride), bacteriostatic water, Ringer's solution, glucose orsaccharose solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions (see for example the most current edition ofRemington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott,Williams & Wilkins). The pH of the composition of the disclosure issuitably adjusted and buffered in order to be appropriate for use inhumans or animals, typically at a physiological or slightly basic pH(between about pH 8 to about pH 9, with a special preference for pH8.5). Suitable buffers include phosphate buffer (e.g. PBS), bicarbonatebuffer and/or Tris buffer. A typical composition is formulated in 1Msaccharose, 150 mM NaCl, 1 mM MgCl2, 54 mg/1 Tween 80, 10 mM Tris pH8.5. Another typical composition is formulated in 10 mg/ml mannitol, 1mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.

The composition of the disclosure can be in various forms, e.g. in solid(e.g. powder, lyophilized form), or liquid (e.g. aqueous). In the caseof solid compositions, the typical methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active agent plusany additional desired ingredient from a previously sterile-filteredsolution thereof. Such solutions can, if desired, be stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection.

Nebulized or aerosolized formulations also form part of this disclosure.Methods of intranasal administration are well known in the art,including the administration of a droplet, spray, or dry powdered formof the composition into the nasopharynx of the individual to be treatedfrom a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer (see forexample WO 95/11664). Enteric formulations such as gastroresistantcapsules and granules for oral administration, suppositories for rectalor vaginal administration also form part of this disclosure. Fornon-parental administration, the compositions can also includeabsorption enhancers which increase the pore size of the mucosalmembrane. Such absorption enhancers include sodium deoxycholate, sodiumglycocholate, dimethyl-beta-cyclodextrin,lauroyl-1-lysophosphatidylcholine and other substances having structuralsimilarities to the phospholipid domains of the mucosal membrane.

The composition can also contain other pharmaceutically acceptableexcipients for providing desirable pharmaceutical or pharmacodynamicproperties, including for example modifying or maintaining the pH,osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution of the formulation, modifying or maintaining release orabsorption into an the human or animal organism. For example, polymerssuch as polyethylene glycol may be used to obtain desirable propertiesof solubility, stability, half-life and other pharmaceuticallyadvantageous properties (Davis et al., 1978, Enzyme Eng. 4, 169-173;Burnham et al., 1994, Am. J. Hosp. Pharm. 51, 210-218). Representativeexamples of stabilizing components include polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Otherstabilizing components especially suitable in plasmid-based compositionsinclude hyaluronidase (which is thought to destabilize the extracellular matrix of the host cells as described in WO 98/53853),chloroquine, protic compounds such as propylene glycol, polyethyleneglycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or derivativesthereof, aprotic compounds such as dimethylsulfoxide (DMSO),diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane,dimethyl-formamide, dimethylacetamide, tetramethylurea, acetonitrile(see EP 890 362), nuclease inhibitors such as actin G (WO 99/56784) andcationic salts such as magnesium (Mg²⁺) (EP 998 945) and lithium (Li⁺)(WO 01/47563) and any of their derivatives. The amount of cationic saltin the composition of the disclosure typically ranges from about 0.1 mMto about 100 mM, and still more typically from about 0.1 mM to about 10mM. Viscosity enhancing agents include sodium carboxymethylcellulose,sorbitol, and dextran. The composition can also contain substances knownin the art to promote penetration or transport across the blood barrieror membrane of a particular organ (e.g. antibody to transferrinreceptor; Friden et al., 1993, Science 259, 373-377). A gel complex ofpoly-lysine and lactose (Midoux et al., 1993, Nucleic Acid Res. 21,871-878) or poloxamer 407 (Pastore, 1994, Circulation 90, 1-517) can beused to facilitate administration in arterial cells.

The composition of the disclosure may also comprise one or moreadjuvant(s) suitable for systemic or mucosal application in humans.Representative examples of useful adjuvants include without limitationalum, mineral oil emulsion such as Freunds complete and incomplete,lipopolysaccharide or a derivative thereof (Ribi et al., 1986,Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ.Corp., NY, p407-419), saponins such as QS21 (Sumino et al., 1998, J.Virol. 72, 4931-4939; WO 98/56415), Escin, Digitonin, Gypsophila orChenopodium quinoa saponins and CpG oligodeoxynucleotides. Alternativelythe composition of the disclosure may be formulated with conventionalvaccine vehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, and lipid-based particles, etc.The composition may also be formulated in the presence of cholesterol toform particulate structures such as liposomes.

The composition may be administered to patients in an amount effective,especially to enhance an immune response in an animal or human organism.As used herein, the term “effective amount” refers to an amountsufficient to realize a desired biological effect. For example, aneffective amount for enhancing an immune response could be that amountnecessary to cause activation of the immune system, for instanceresulting in the development of an anti-tumor response in a cancerouspatient (e.g. size reduction or regression of the tumor into which thecomposition has been injected and/or distant tumors). The appropriatedosage may vary depending upon known factors such as the pharmacodynamiccharacteristics of the particular active agent, age, health, and weightof the host organism; the condition(s) to be treated, nature and extentof symptoms, kind of concurrent treatment, frequency of treatment, theneed for prevention or therapy and/or the effect desired. The dosagewill also be calculated dependent upon the particular route ofadministration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by a practitioner, in the light of the relevant circumstances. Thetiter may be determined by conventional techniques. A composition basedon vector plasmids may be formulated in the form of doses of between 1μg to 100 mg, advantageously between 10 μg and 10 mg and typicallybetween 100 μg and 1 mg. A composition based on proteins may beformulated in the form of doses of between 10 ng to 100 mg. A typicaldose is from about 1 μg to about 10 mg of the therapeutic protein per kgbody weight. The administration may take place in a single dose or adose repeated one or several times after a certain time interval. In onetypical embodiment, the composition of the present disclosure isadministered by injection using conventional syringes and needles, ordevices designed for ballistic delivery of solid compositions (WO99/27961), or needleless pressure liquid jet device (U.S. Pat. No.4,596,556; U.S. Pat. No. 5,993,412).

The composition of the disclosure can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic. Inall cases, the composition must be sterile and should be fluid to theextent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.Sterile injectable solutions can be prepared by incorporating the activeagent (e.g., a fusion protein or infectious particles) in the requiredamount with one or a combination of ingredients enumerated above,followed by filtered sterilization.

Methods of Use

The pharmaceutical composition of the disclosure may be employed inmethods for treating or preventing a variety of diseases and pathologicconditions, including genetic diseases, congenital diseases and acquireddiseases such as infectious diseases (e.g. viral and/or bacterialinfections), cancer, immune deficiency diseases, and autoimmunediseases. Accordingly, the present disclosure also encompasses the useof the fusion protein, vector, infectious viral particle, host cell orcomposition of the disclosure for the preparation of a drug intended fortreating or preventing such diseases, and especially cancer or aninfectious disease.

The composition of the present disclosure is particularly intended forthe preventive or curative treatment of disorders, conditions ordiseases associated with cancer. The term “cancer” encompasses anycancerous conditions including diffuse or localized tumors, metastasis,cancerous polyps and preneoplastic lesions (e.g. dysplasies) as well asdiseases which result from unwanted cell proliferation. A variety oftumors may be selected for treatment in accordance with the methodsdescribed herein. In general, solid tumors are typical. Cancers whichare contemplated in the context of the disclosure include withoutlimitation glioblastoma, sarcoma, melanomas, mastocytoma, carcinomas aswell as breast cancer, prostate cancer, testicular cancer, ovariancancer, endometrial cancer, cervical cancer (in particular, thoseinduced by a papilloma virus), lung cancer (e.g. lung carcinomasincluding large cell, small cell, squamous and adeno-carcinomas), renalcancer, bladder cancer, liver cancer, colon cancer, anal cancer,pancreatic cancer, stomach cancer, gastrointestinal cancer, cancer ofthe oral cavity, larynx cancer, brain and CNS cancer, skin cancer (e.g.melanoma and non-melanoma), blood cancer (lymphomas, leukemia,especially if they have developed in solid mass), bone cancer,retinoblastoma and thyroid cancer. In one typical embodiment of the useof the disclosure, the composition is administered into or in closeproximity to a solid tumor.

In certain embodiments, the disclosure contemplates uses of conjugatesdisclosed herein in autologous immune enhancement therapy (AIET). AIETis a treatment method in which immune or cancer cells, e.g.,lymphokine-activated killer (LAK) cells, natural killer (NK) cells,cytotoxic T lymphocytes (CTLs), dendritic cells (DCs), are taken outfrom the body of a subject which are cultured and processed to activatethem until their resistance to cancer is strengthened and then the cellsare put back in the body. The cells, antibodies, and organs of theimmune system work to protect and defend the body against the tumorcells. In certain embodiments, the disclosure contemplates mixingharvested cells with conjugates of GM-CSF and IL-4 to activate them. Incertain embodiments, the disclosure contemplates administeringconjugates of GM-CSF and IL-4 when the cells are administered back tothe subject.

In certain embodiments, the disclosure contemplates the administrationof sipuleucel-T (PROVENGE) in combination with a conjugate of GM-CSF andIL-4. PROVENGE consists of autologous peripheral blood mononuclearcells, including antigen presenting cells (APCs), that have beenactivated during a culture period with a recombinant human protein,PAP-GM-CSF, consisting of prostatic acid phosphatase (PAP), an antigenexpressed in prostate cancer tissue, linked to GM-CSF. In certainembodiments, the disclosure relates to a conjugate comprising PAP,GM-CSF, and IL-4, and uses in activating antigen presenting cells inperipheral blood mononuclear cells. The peripheral blood mononuclearcells of the subject may be obtained via a standard leukapheresisprocedure prior to infusion. During culture, the recombinant antigen canbind to and be processed by antigen presenting cells (APCs). Therecombinant antigen is believed to direct the immune response to PAP.The infused product is believed to contain antigen presenting cells,dendritic cells, T cells, B cells, natural killer (NK) cells, and othercells. Typically each dose contains more than 50 million autologousCD54⁺ cells activated with PAP-GM-CSF or PAP-GM-CSF-IL-4. The potency istypically evaluated by measuring the increased expression of the CD54molecule, also known as ICAM-1, on the surface of APCs after culturewith PAP-GM-CSF or PAP-CM-CSF-IL-4. CD54 is a cell surface molecule thatplays a role in the immunologic interactions between APCs and T cells,and is considered a marker of immune cell activation.

In certain embodiments, the disclosure contemplates methods for treatingcancer comprising administering any GM-CSF and IL-4 conjugate disclosedherein as an immune adjuvant in combination with a vector that encodes atumor associated antigen/cancer marker, such as PSA, PAP, and optionallyencoding other co-stimulatory molecules selected from, B7-1, B7-2,ICAM-1, GM-CSF, leukocyte function-associated antigen-3 (LFA-3). Otherembodiments contemplated for the treatment of cancer includeadministering an effective amount of a vector that encodes a GM-CSF andIL-4 conjugate disclosed herein and optionally further encodes a tumorassociated antigen/cancer marker and optionally encodes otherco-stimulatory molecules to a subject. PROSTVAC is a recombinant vectorencoding costimulatory molecules, as well as PSA as a vaccine target.Plasmid DNA is incorporated into either vaccinia or fowlpox viruses bymeans of a packing cell line. Patients are treated with a vaccinia primefollowed by a series of fowlpox-based boosts.

In certain embodiments, the disclosure relates to methods of treatingcancer comprising administering a GM-CSF and IL-4 conjugate incombination with an anti-CTLA-4 antibody. Anti-CTLA-4 antibody iscontemplated to be administered in combination with any of the methodsdisclosed herein. It is believed that it binds to CTLA-4 surfaceglycoprotein on T-cell surface, minimizing immune autoregulation andpotentially enhancing antitumor activity. Interactions between B7molecules on antigen-presenting cells and CTLA-4 on tumor-specific Tcells are inhibitory. Thus, CTLA-4 engagement negatively regulates theproliferation and function of such T cells. Under certain conditions,blocking CTLA-4 with a monoclonal antibody (ipilimumab or tremilimumab)restores T-cell function.

Other embodiments contemplated for the treatment of cancer includemethods that utilize the extraction of cancer cells from a subject andincorporate glycosyl-phosphatidylinositol (GPI)-anchored co-stimulatorymolecules such as B7-1 and B7-2 into tumor cell membranes optionallywith a conjugate GM-CSF and IL-4 anchored GPI, and administering themodified cells to the subject in combination with a conjugate of GM-CSFand IL-7 to elicit an immune response. See e.g., McHugh et al., CancerRes., 1999, 59(10):2433-7; Poloso et al., Mol Immunol., 2002,38(11):803-16; and Nagarajan & Selvaraj, Cancer Res., 2002,62(10):2869-74.

Other pathologic diseases and conditions are also contemplated in thecontext of the disclosure, especially infectious diseases associatedwith an infection by a pathogen such as fungi, bacteria, protozoa andviruses. Representative examples of viral pathogens include withoutlimitation human immunodeficiency virus (e.g. HIV-1 or HIV-2), humanherpes viruses (e.g. HSV1 or HSV2), cytomegalovirus, Rotavirus, EpsteinBarr virus (EBV), hepatitis virus (e.g. hepatitis B virus, hepatitis Avirus, hepatitis C virus and hepatitis E virus), varicella-zoster virus(VZV), paramyxoviruses, coronaviruses; respiratory syncytial virus,parainfluenza virus, measles virus, mumps virus, flaviviruses (e.g.Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus), influenza virus, and typically humanpapilloma viruses (e.g. HPV-6, 11, 16, 18, 31. 33). Representativeexamples of bacterial pathogens include Neisseria (e.g. N. gonorrhea andN. meningitidis); Bordetella (e.g. B. pertussis, B. parapertussis and B.bronchiseptica), Mycobacteria (e.g. M. tuberculosis, M. bovis, M.leprae, M. avium, M. paratuberculosis, M. smegmatis); Legionella (e.g.L. pneumophila); Escherichia (e.g. enterotoxic E. coli, enterohemorragicE. coli, enteropathogenic E. coli); Vibrio (e.g. V. cholera); Shigella(e.g. S. sonnei, S. dysenteriae, S. flexnerii); Salmonella (e.g. S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis); Listeria (e.g. L.monocytogenes); Helicobacter (e.g. H. pylori); Pseudomonas (e.g. P.aeruginosa); Staphylococcus (e.g. S. aureus, S. epidermidis);Enterococcus (e.g. E. faecalis, E. faecium), Clostridium (e.g. C.tetani, C. botulinum, C. difficile); Bacillus (e.g. B. anthracis);Corynebacterium (e.g. C. diphtheriae), and Chlamydia (e.g. C.trachomatis, C. pneumoniae, C. psittaci). Representative examples ofparasite pathogens include Plasmodium (e.g. P. falciparum), Toxoplasma(e.g. T. gondii) Leshmania (e.g. L. major), Pneumocystis (e.g. P.carinii), Trichomonas (e.g. T. vaginalis), Schisostoma (e.g. S.mansoni). Representative examples of fungi include Candida (e.g. C.albicans) and Aspergillus.

Examples of autoimmune diseases include, but are not limited to,multiple sclerosis (MS), scleroderma, rheumatoid arthritis, autoimmunehepatitis, diabetes mellitus, ulcerative colitis, Myasthenia gravis,systemic lupus erythematosus, Graves' disease, idiopathicthrombocytopenia purpura, hemolytic anemia, multiplemyositis/dermatomyositis, Hashimoto's disease, autoimmune hypocytosis,Sjogren's syndrome, angitis syndrome and drug-induced autoimmunediseases (e.g., drug-induced lupus).

Moreover, as mentioned above, the fusion protein, nucleic acid molecule,vector, infectious particle, host cell and/or composition of the presentdisclosure can be used as an adjuvant to enhance the immune response ofan animal or human organism to a particular antigen. This particular useof the present disclosure may be made in combination with one or moretransgenes or transgene products as defined above, e.g. for purposes ofimmunotherapy. Typically, the active agent (e.g. fusion protein,infectious particle or pharmaceutical composition of the disclosure) isadministered in combination with one or more transgenes or transgeneproducts. Accordingly, there is typically also provided a compositioncomprising in combination a transgene product (e.g. a viral antigen or asuicide gene product) and a fusion protein as well as a compositioncomprising vector(s) or viral particles encoding a transgene product anda fusion protein. The transgene and the fusion-encoding nucleic acidsequences may be expressed from the same vector or from separate vectorswhich may have the same origin (e.g. adenoviral vectors) or a differentorigin (e.g. a MVA vector encoding the particular antigen and anadenoviral vector encoding the fusion protein). The fusion protein andthe transgene product (or their respective encoding vectors) can beintroduced into the host cell or organism either concomitantly orsequentially either via the mucosal and/or systemic route.

Combination Therapies

The cancer treatments disclosed herein can be applied as a sole therapyor can involve, conventional surgery or radiotherapy, hormonal therapy,or chemotherapy. Such chemotherapy can include one or more of thefollowing categories of anti-tumour agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan,chlorambucil, busulfan and nitrosoureas); antimetabolites (for exampleantifolates such as fluoropyrimidines like 5-fluorouracil andgemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinosideand hydroxyurea); antitumour antibiotics (for example anthracyclineslike adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin,idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitoticagents (for example vinca alkaloids like vincristine, vinblastine,vindesine and vinorelbine and taxoids like taxol and taxotere); andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin); and proteosomeinhibitors (for example bortezomib [Velcade®]); and the agent anegrilide[Agrylin®]; and the agent alpha-interferon

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), LHRHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane) and inhibitors of 5α-reductase such as finasteride;

(iii) agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-Her2 antibody trastuzumab and the anti-epidermal growthfactor receptor (EGFR) antibody, cetuximab), farnesyl transferaseinhibitors, tyrosine kinase inhibitors and serine/threonine kinaseinhibitors, for example inhibitors of the epidermal growth factor familyfor example EGFR family tyrosine kinase inhibitors such as:N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib), and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine(CI 1033), for example inhibitors of the platelet-derived growth factorfamily and for example inhibitors of the hepatocyte growth factorfamily, for example inhibitors of phosphotidylinositol 3-kinase (PI3K)and for example inhibitors of mitogen activated protein kinase kinase(MEK1/2) and for example inhibitors of protein kinase B (PKB/Akt), forexample inhibitors of Src tyrosine kinase family and/or Abelson (AbI)tyrosine kinase family such as dasatinib (BMS-354825) and imatinibmesylate (Gleevec™); and any agents that modify STAT signalling;

(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™]) andcompounds that work by other mechanisms (for example linomide,inhibitors of integrin ocvβ3 function and angiostatin);

(vi) vascular damaging agents such as Combretastatin A4;

(vii) antisense therapies, for example those which are directed to thetargets listed above, such as an anti-RAS antisense; and

(viii) immunotherapy approaches, including for example ex-vivo andin-vivo approaches to increase the immunogenicity of subject tumourcells, such as transfection with cytokines such as interleukin 2,interleukin 4 or granulocyte-macrophage colony stimulating factor,approaches to decrease T-cell energy, approaches using transfectedimmune cells such as cytokine-transfected dendritic cells, approachesusing cytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies, and approaches using the immunomodulatorydrugs thalidomide and lenalidomide [Revlimid®].

The combination therapy also contemplates use of the disclosedpharmaceutical compositions with radiation therapy or surgery, as analternative, or a supplement, to a second therapeutic orchemotherapeutic agent.

A typical chronic lymphocytic leukemia (CLL) chemotherapeutic planincludes combination chemotherapy with chlorambucil or cyclophosphamide,plus a corticosteroid such as prednisone or prednisolone. The use of acorticosteroid has the additional benefit of suppressing some relatedautoimmune diseases, such as immunohemolytic anemia or immune-mediatedthrombocytopenia. In resistant cases, single-agent treatments withnucleoside drugs such as fludarabine, pentostatin, or cladribine may besuccessful. Patients may consider allogeneic or autologous bone marrowtransplantation. In certain embodiments, the disclosure contemplatescombination treatments using conjugates disclosed herein in combinationwith chloroambucil, cyclophosphamide, prednisone, prednisolone,fludarabine, pentostatin, and/or cladribine or combinations thereof.

Treatment of acute lymphoblastic leukemia typically includeschemotherapy to bring about bone marrow remission. Typical regimentsinclude prednisone, vincristine, and an anthracycline drug,L-asparaginase or cyclophosphamide. Other options include tprednisone,L-asparaginase, and vincristine. Consolidation therapy orintensification therapy to eliminate any remaining leukemia may includeantimetabolite drugs such as methotrexate and 6-mercaptopurine (6-MP).In certain embodiments, the disclosure contemplates combinationtreatments using conjugates disclosed herein in combination with COP,CHOP, R-CHOP, imatinib, alemtuzumab, vincristine, L-asparaginase orcyclophosphamide, methotrexate and/or 6-mercaptopurine (6-MP). COPrefers to a chemotherapy regimen used in the treatment of lymphoma ofcyclophosphamide, vincristine, and prednisone or prednisolone andoptionally hydroxydaunorubicin (CHOP) and optionally rituximab (R-CHOP).

In some embodiments, the disclosure relates to treating a viralinfection by administering a GM-CSF and IL-4 conjugate in combinationwith a second antiviral agent. In further embodiments, a GM-CSF and IL-4conjugate is administered in combination with one or more of thefollowing agents: abacavir, acyclovir, acyclovir, adefovir, amantadine,amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir,cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol,edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, interferon type III, interferon type II, interferon type I,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, oseltamivir (Tamiflu), peginterferonalfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin,raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir,stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine,trizivir, tromantadine, truvada, valaciclovir (Valtrex), valganciclovir,vicriviroc, vidarabine, viramidine zalcitabine, zanamivir (Relenza),and/or zidovudine (AZT).

Antiviral agents include, but are not limited to, protease inhibitors(PIs), integrase inhibitors, entry inhibitors (fusion inhibitors),maturation inhibitors, and reverse transcriptase inhibitors(anti-retrovirals). Combinations of antiviral agents create multipleobstacles to viral replication, i.e., to keep the number of offspringlow and reduce the possibility of a superior mutation. If a mutationthat conveys resistance to one of the agents being taken arises, theother agents continue to suppress reproduction of that mutation. Forexample, a single anti-retroviral agent has not been demonstrated tosuppress an HIV infection for long. These agents are typically taken incombinations in order to have a lasting effect. As a result, thestandard of care is to use combinations of anti-retrovirals.

Reverse transcribing viruses replicate using reverse transcription,i.e., the formation of DNA from an RNA template. Retroviruses oftenintegrate the DNA produced by reverse transcription into the hostgenome. They are susceptible to antiviral drugs that inhibit the reversetranscriptase enzyme. In certain embodiments the disclosure relates tomethods of treating viral infections by administering a GM-CSF and IL-4conjugate, and a retroviral agent such as nucleoside and nucleotidereverse transcriptase inhibitors (NRTI) and/or a non-nucleoside reversetranscriptase inhibitors (NNRTI). Examples of nucleoside reversetranscriptase inhibitors include zidovudine, didanosine, zalcitabine,stavudine, lamivudine, abacavir, emtricitabine, entecavir, apricitabine.Examples of nucleotide reverse transcriptase inhibitors includetenofovir and adefovir. Examples of non-nucleoside reverse transcriptaseinhibitors include efavirenz, nevirapine, delavirdine, and etravirine.

In certain embodiments, the disclosure relates to methods of treating aviral infection by administering a GM-CSF and IL-4 conjugate optionallywith an antigen in combination with an antiviral drug, e.g.,2′,3′-dideoxyinosine and a cytostatic agent, e.g., hydroxyurea.

Human immunoglobulin G (IgG) antibodies are believed to have opsonizingand neutralizing effects against certain viruses. IgG is sometimesadministered to a subject diagnosed with immune thrombocytopenic purpura(ITP) secondary to a viral infection since certain viruses such as, HIVand hepatitis, cause ITP. In certain embodiments, the disclosure relatesto methods of treating or preventing viral infections comprisingadministering a GM-CSF and IL-4 conjugate in combination with animmunoglobulin to a subject. IgG is typically manufactured from largepools of human plasma that are screened to reduce the risk of undesiredvirus transmission. The Fc and Fab functions of the IgG molecule areusually retained. Therapeutic IgGs include Privigen, Hizentra, andWinRho. WinRho is an immunoglobulin (IgG) fraction containing antibodiesto the Rho(D) antigen (D antigen). The antibodies have been shown toincrease platelet counts in Rho(D) positive subjects with ITP. Themechanism is thought to be due to the formation of anti-Rho(D)(anti-D)-coated RBC complexes resulting in Fc receptor blockade, thussparing antibody-coated platelets.

In some embodiments, the disclosure relates to treating a bacterialinfection by administering a GM-CSF and IL-4 conjugate in combinationwith an antibiotic drug. In further embodiments, the subject isco-administered with an antibiotic selected from the group comprising ofSulfonamides, Diaminopyrimidines, Quinolones, Beta-lactam antibiotics,Cephalosporins, Tetracyclines, Notribenzene derivatives,Aminoglycosides, Macrolide antibiotics, Polypeptide antibiotics,Nitrofuran derivatives, Nitroimidazoles, Nicotinin acid derivatives,Polyene antibiotics, Imidazole derivatives or Glycopeptide, Cycliclipopeptides, Glycylcyclines and Oxazolidinones. In further embodiments,these antibiotics include but are not limited to Sulphadiazine,Sulfones—[Dapsone (DDS) and Paraaminosalicyclic (PAS)], Sulfanilamide,Sulfamethizole, Sulfamethoxazole, Sulfapyridine, Trimethoprim,Pyrimethamine, Nalidixic acids, Norfloxacin, Ciproflaxin, Cinoxacin,Enoxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Ofloxacin, Pefloxacin, Sparfloxacin,Trovafloxacin, Penicillins (Amoxicillin, Ampicillin, Azlocillin,Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Hetacillin,Oxacillin, Mezlocillin, Penicillin G, Penicillin V, Piperacillin),Cephalosporins (Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin,Cefalonium, Cefaloridin, Cefalotin, Cefapirin, Cefatrizine, Cefazaflur,Cefazedone, Cefazolin, Cefradine, Cefroxadine, Ceftezole, Cefaclor,Cefonicid, Ceforanide, Cefprozil, Cefuroxime, Cefuzonam, Cefmetazole,Cefoteta, Cefoxitin, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren,Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefoperazone, Cefotaxime,Cefotiam, Cefpimizole, Cefpiramide, Cefpodoxime, Cefteram, Ceftibuten,Ceftiofur, Ceftiolen, Ceftizoxime, Ceftriaxone, Cefoperazone,Ceftazidime, Cefepime), Moxolactam, Carbapenems (Imipenem, Ertapenem,Meropenem), Monobactams (Aztreonam), Oxytetracycline, Chlortetracycline,Clomocycline, Demeclocycline, Tetracycline, Doxycycline, Lymecycline,Meclocycline, Methacycline, Minocycline, Rolitetracycline,Chloramphenicol, Amikacin, Gentamicin, Framycetin, Kanamycin, Neomicin,Neomycin, Netilmicin, Streptomycin, Tobramycin, Azithromycin,Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,Telithromycin, Polymyxin-B, Colistin, Bacitracin, TyrothricinNotrifurantoin, Furazolidone, Metronidazole, Tinidazole, Isoniazid,Pyrazinamide, Ethionamide, Nystatin, Amphotericin-B, Hamycin,Miconazole, Clotrimazole, Ketoconazole, Fluconazole, Rifampacin,Lincomycin, Clindamycin, Spectinomycin, Chloramphenicol, Clindamycin,Colistin, Fosfomycin, Loracarbef, Metronidazole, Nitrofurantoin,Polymyxin B, Polymyxin B Sulfate, Procain, Spectinomycin, Tinidazole,Trimethoprim, Ramoplanin, Teicoplanin, Vancomycin, Trimethoprim,Sulfamethoxazole, and/or Nitrofurantoin.

Vectors

The term “vector” as used herein refers to both expression andnon-expression vectors and includes viral as well as non-viral vectors,including autonomous self-replicating circular plasmids. Where arecombinant microorganism or cell culture is described as hosting an“expression vector,” this includes both extra-chromosomal circular DNAand DNA that has been incorporated into the host chromosome(s). Typicalvectors of the disclosure are expression vectors. An expression vectorcontains multiple genetic elements positionally and sequentiallyoriented, i.e., operatively linked with other necessary elements suchthat nucleic acid molecule in the vector encoding the fusion proteins ofthe disclosure can be transcribed, and when necessary, translated in thehost cells.

Any type of vector can be used in the context of the present disclosure,whether of plasmid or viral origin, whether it is an integrating ornon-integrating vector. Such vectors are commercially available ordescribed in the literature. Contemplated in the context of thedisclosure are vectors for use in gene therapy (i.e. which are capableof delivering the nucleic acid molecule to a target cell) as well asexpression vectors for use in recombinant techniques (i.e. which arecapable for example of expressing the nucleic acid molecule of thedisclosure in cultured host cells).

The vectors of the disclosure can function in prokaryotic or eukaryoticcells or in both (shuttle vectors). Suitable vectors include withoutlimitation vectors derived from bacterial plasmids, bacteriophages,yeast episomes, artificial chromosomes, such as BAC, PAC, YAC, or MAC,and vectors derived from viruses such as baculoviruses, papovaviruses(e.g. SV40), herpes viruses, adenoviruses, adenovirus-associated viruses(AAV), poxviruses, foamy viruses, and retroviruses. Vectors may also bederived from combinations of these sources such as those derived fromplasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.Viral vectors can be replication-competent, conditionally replicative orreplication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Examples of suitable plasmids include but are not limited to thosederived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript(Stratagene), p Poly (Lathe et al., 1987, Gene 57, 193-201), pTrc (Amannet al., 1988, Gene 69, 301-315) and pET 11d (Studier et al., 1990, GeneExpression Technology: Methods in Enzymology 185, 60-89). It is wellknown that the four of the plasmid can affect the expression efficiency,and it is typical that a large fraction of the vector be in supercoiledform. Examples of vectors for expression in yeast (e.g. S. cerevisiae)include pYepSec1 (Baldari et al., 1987, EMBO J. 6, 229-234), pMFa (Kujanet al., 1982, Cell 30, 933-943), pJRY88 (Schultz et al., 1987, Gene 54,113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Thevectors of the disclosure can also be derived from baculoviruses to beexpressed in cultured insect cells (e.g. Sf 9 cells).

According to a typical embodiment of the disclosure, the nucleic acidmolecules described herein are expressed by using mammalian expressionvectors. Examples of mammalian expression vectors include pREP4, pCEP4(Invitrogene), pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840) andpMT2PC (Kaufman et al., 1987, EMBO J. 6, 187-195). The expressionvectors listed herein are provided by way of example only of somewell-known vectors available to those of ordinary skill in the art. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance, propagation or expression of the nucleic acidmolecules described herein.

Moreover, the vector of the present disclosure may also comprise amarker gene in order to select or to identify the transfected cells(e.g. by complementation of a cell auxotrophy or by antibioticresistance), stabilizing elements (e.g. cer sequence; Summers andSherrat, 1984, Cell 36, 1097-1103), integrative elements (e.g. LTR viralsequences and transposons) as well as elements providing aself-replicating function and enabling the vector to be stablymaintained in cells, independently of the copy number of the vector inthe cell. Markers include tetracycline or ampicillin-resistance genesfor prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective. The self-replicatingfunction may be provided by using a viral origin of replication andproviding one or more viral replication factors that are required forreplication mediated by that particular viral origin (WO 95/32299).Origins of replication and any replication factors may be obtained froma variety of viruses, including Epstein-Barr virus (EBV), human andbovine papilloma viruses and papovavirus BK.

Typical vectors of the present disclosure are viral vectors andespecially adenoviral vectors, which have a number of well-documentedadvantages as vectors for gene therapy. The adenoviral genome consistsof a linear double-stranded DNA molecule of approximately 36 kb carryingmore than about thirty genes necessary to complete the viral cycle. Theearly genes are divided into 4 regions (E1 to E4) that are essential forviral replication (Pettersson and Roberts, 1986, In Cancer Cells (Vol4): DNA Tumor Viruses, Botchan and Glodzicker Sharp Eds pp 37-47, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Halbert et al.,1985, J. Virol. 56, 250-257) with the exception of the E3 region, whichis believed dispensable for viral replication based on the observationthat naturally-occurring mutants or hybrid viruses deleted within the E3region still replicate like wild-type viruses in cultured cells (Kellyand Lewis, 1973, J. Virol. 12, 643-652). The E1 gene products encodeproteins responsible for the regulation of transcription of the viralgenome. The E2 gene products are required for initiation and chainelongation in viral DNA synthesis. The proteins encoded by the E3prevent cytolysis by cytotoxic T cells and tumor necrosis factor (Woldand Gooding, 1991, Virology 184, 1-8). The proteins encoded by the E4region are involved in DNA replication, late gene expression andsplicing and host cell shut off (Halbert et al., 1985, J. Virol. 56,250-257). The late genes (L1 to L5) encode in their majority thestructural proteins constituting the viral capsid. They overlap at leastin part with the early transcription units and are transcribed from aunique promoter (MLP for Major Late Promoter). In addition, theadenoviral genome carries at both extremities cis-acting 5′ and 3′ ITRs(Inverted Terminal Repeat) and the encapsidation region, both essentialfor DNA replication. The ITRs harbor origins of DNA replication whereasthe encapsidation region is required for the packaging of adenoviral DNAinto infectious particles.

The adenoviral vectors for use in accordance with the presentdisclosure, typically infects mammalian cells. It can be derived fromany human or animal source, in particular canine (e.g. CAV-1 or CAV-2;Genbank ref CAV1GENOM and CAV77082 respectively), avian (Genbank refAAVEDSDNA), bovine (such as BAV3; Seshidhar Reddy et al., 1998, J.Virol. 72, 1394-1402), murine (Genbank ref ADRMUSMAV1), ovine, feline,porcine or simian adenovirus or alternatively from a hybrid thereof. Anyserotype can be employed from the adenovirus serotypes 1 through 51. Forinstance, an adenovirus can be of subgroup A (e.g. serotypes 12, 18, and31), subgroup B (e.g. serotypes 3, 7, 11, 14, 16, 21, 34, and 35),subgroup C (e.g. serotypes 1, 2, 5, and 6), subgroup D (e.g. serotypes8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroupE (serotype 4), subgroup F (serotypes 40 and 41), or any otheradenoviral serotype. However, the human adenoviruses of the B or Csub-group are typical and especially adenoviruses 2 (Ad2), 5 (Ad5) and35 (Ad35). Generally speaking, adenoviral stocks that can be employed asa source of the cited adenovirus are currently available from theAmerican Type Culture Collection (ATCC, Rockville, Md.), or from anyother source. Moreover, such adenoviruses have been the subject ofnumerous publications describing their sequence, organization andbiology, allowing the artisan to apply them. Adenoviral vectors, methodsof producing adenoviral vectors, and methods of using adenoviral vectorsare disclosed, for example in U.S. Pat. No. 6,133,028, U.S. Pat. No.6,040,174, U.S. Pat. No. 6,110,735, U.S. Pat. No. 6,399,587, WO 00/50573and EP 1016711 for group C adenoviral vectors and for example in U.S.Pat. No. 6,492,169 and WO 02/40665 for non-group C adenoviral vectors.

In certain embodiments, the adenoviral vector of the present disclosureis replication-competent. The term “replication-competent” as usedherein refers to an adenoviral vector capable of replicating in a hostcell in the absence of any trans-complementation. In the context of thepresent disclosure, this term also encompasses replication-selective orconditionally-replicative adenoviral vectors which are engineered toreplicate better or selectively in cancer or hyperproliferative hostcells. Examples of such replication-competent adenoviral vectors arewell known in the art and readily available to those skill in the art(see, for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11,2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany etal., 2000, Nature Biotechnology 18, 723-727).

Replication-competent adenoviral vectors according to the disclosure canbe a wild-type adenovirus genome or can be derived therefrom byintroducing modifications into the viral genome, e.g., for the purposeof generating a conditionally-replicative adenoviral vector. Suchmodification(s) include the deletion, insertion and/or mutation of oneor more nucleotide(s) in the coding sequences and/or the regulatorysequences. Typical modifications are those that render saidreplication-competent adenoviral vector dependent on cellular activitiesspecifically present in a tumor or cancerous cell. In this regard, viralgene(s) that become dispensable in tumor cells, such as the genesresponsible for activating the cell cycle through p53 or Rb binding, canbe completely or partially deleted or mutated. By way of illustration,such conditionally-replicative adenoviral vectors can be engineered bythe complete deletion of the adenoviral MB gene encoding the 55 kDaprotein or the complete deletion of the MB region to abrogate p53binding (see for example U.S. Pat. No. 5,801,029 and U.S. Pat. No.5,846,945). This prevents the virus from inactivating tumor suppressionin normal cells, which means that the virus cannot replicate. However,the virus will replicate and lyse cells that have shut off p53 or Rbexpression through oncogenic transformation. As another example, thecomplete deletion of the E1A region makes the adenoviral vectordependent on intrinsic or IL-6-induced E1A-like activities. Optionally,an inactivating mutation may also be introduced in the E1A region toabrogate binding to the Rb. Rb defective mutation/deletion is typicallyintroduced within the E1A CR1 and/or CR2 domain (see for exampleWO00/24408). In a second strategy optionally or in combination to thefirst approach, native viral promoters controlling transcription of theviral genes can be replaced with tissue or tumor-specific promoters. Byway of illustration, regulation of the E1A and/or the E1B genes can beplaced under the control of a tumor-specific promoter such as the PSA,the kallikrein, the probasin, the AFP, the a-fetoprotein or thetelomerase reverse transcriptase (TERT) promoter (see for example U.S.Pat. No. 5,998,205, WO 99/25860, U.S. Pat. No. 5,698,443 and WO00/46355) or a cell-cycle specific promoter such as E2F-1 promoter(WO00/15820 and WO01/36650). Typical in this context is the exemplaryvector designated ONYX-411 which combines a Rb defective deletion of 8amino acid residues within the MA CR2 domain and the use of E2F-1promoter to control expression of both the E1A and E4 viral genes.

In certain embodiments, the adenoviral vector of the disclosure isreplication-defective. Replication-defective adenoviral vectors areknown in the art and can be defined as being deficient in one or moreregions of the adenoviral genome that are essential to the viralreplication (e.g., E1, E2 or E4 or combination thereof), and thus unableto propagate in the absence of trans-complementation (e.g., provided byeither complementing cells or a helper virus). The replication-defectivephenotype is obtained by introducing modifications in the viral genometo abrogate the function of one or more viral gene(s) essential to theviral replication. Typical replication-defective vectors are E1-deleted,and thus defective in E1 function. Such E1-deleted adenoviral vectorsinclude those described in U.S. Pat. No. 6,063,622; U.S. Pat. No.6,093,567; WO 94/28152; WO 98/55639 and EP 974 668 (the disclosures ofall of these publications are hereby incorporated herein by reference).A typical E1 deletion covers approximately the nucleotides (nt) 459 to3328 or 459 to 3510, by reference to the sequence of the humanadenovirus type 5 (disclosed in the Genbank under the accession number M73260 and in Chroboczek et al., 1992, Virol. 186, 280-285).

Furthermore, the adenoviral backbone of the vector may comprisemodifications (e.g. deletions, insertions or mutations) in additionalviral region(s), to abolish the residual synthesis of the viral antigensand/or to improve long-term expression of the nucleic acid molecules inthe transduced cells (see for example WO 94/28152; Lusky et al., 1998,J. Virol 72, 2022-2032; Yeh et al., 1997, FASEB J. 11, 615-623). In thiscontext, the present disclosure contemplates the use of adenoviralvectors lacking E1, or E1 and E2, or E1 and E3, or E1 and E4, or E1 andE2 and E3, or E1 and E2 and E4, or E1 and E3 and E4, or E1 and E2 and E3and E4 functions. An adenoviral vector defective for E2 function may bedeleted of all or part of the E2 region (typically within the E2A oralternatively within the E2B or within both the E2A and the E2B regions)or comprises one or more mutations, such as the thermosensitive mutationof the DBP (DNA Binding Protein) encoding gene (Ensinger et al., J.Virol. 10 (1972), 328-339). The adenoviral vector may also be deleted ofall or part of the E4 region (see, for example, EP 974 668 and WO00/12741). An exemplary E4 deletion covers approximately the nucleotidesfrom position 32994 to position 34998, by reference to the sequence ofthe human adenovirus type 5. In addition, deletions within thenon-essential E3 region (e.g. from Ad5 position 28597 to position 30469)may increase the cloning capacity, but it may be advantageous to retainthe E3 sequences coding for gp19k, 14.7K and/or RID allowing to escapethe host immune system (Gooding et al., 1990, Critical Review ofImmunology 10, 53-71) and inflammatory reactions (EP 00 440 267.3). Itis also conceivable to employ a minimal (or gutless) adenoviral vectorwhich lacks all functional genes including early (E1, E2, E3 and E4) andlate genes (L1, L2, L3, L4 and L5) with the exception of cis-actingsequences (see for example Kovesdi et al., 1997, Current Opinion inBiotechnology 8, 583-589; Yeh and Perricaudet, 1997, FASEB 11, 615-623;WO 94/12649; and WO 94/28152). The replication-deficient adenoviralvector may be readily engineered by one skilled in the art, taking intoconsideration the required minimum sequences, and is not limited tothese exemplary embodiments.

The nucleic acid molecule of the present disclosure can be inserted inany location of the adenoviral genome, with the exception of thecis-acting sequences. Typically, it is inserted in replacement of adeleted region (E1, E3 and/or E4), with a special preference for thedeleted E1 region. In addition, the expression cassette may bepositioned in sense or antisense orientation relative to the naturaltranscriptional direction of the region in question.

A retroviral vector is also suitable in the context of the presentdisclosure. Retroviruses are a class of integrative viruses whichreplicate using a virus-encoded reverse transcriptase, to replicate theviral RNA genome into double stranded DNA which is integrated intochromosomal DNA of the infected cells. The numerous vectors described inthe literature may be used within the framework of the presentdisclosure and especially those derived from murine leukemia viruses,especially Moloney (Gilboa et al., 1988, Adv. Exp. Med. Biol. 241, 29)or Friend's FB29 strains (WO 95/01447). Generally, a retroviral vectoris deleted of all or part of the viral genes gag, pol and env andretains 5′ and 3′ LTRs and an encapsidation sequence. These elements maybe modified to increase expression level or stability of the retroviralvector. Such modifications include the replacement of the retroviralencapsidation sequence by one of a retrotransposon such as VL30 (U.S.Pat. No. 5,747,323). The nucleic acid molecule of the disclosure can beinserted downstream of the encapsidation sequence, typically in oppositedirection relative to the retroviral genome.

A poxviral vector is also suitable in the context of the presentdisclosure. Poxviruses are a group of complex enveloped viruses thatdistinguish from the above-mentioned viruses by their large DNA genomeand their cytoplasmic site of replication. The genome of several membersof poxyiridae has been mapped and sequenced. It is a double-stranded DNAof approximately 200 kb coding for about 200 proteins of whichapproximately 100 are involved in virus assembly. In the context of thepresent disclosure, a poxyiral vector may be obtained from any member ofthe poxyiridae, in particular canarypox, fowlpox and vaccinia virus, thelatter being typical. Suitable vaccinia viruses include withoutlimitation the Copenhagen strain (Goebel et al., 1990, Virol. 179,247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), theWyeth strain and the modified Ankara (MVA) strain (Antoine et al., 1998,Virol. 244, 365-396). The general conditions for constructing poxviruscomprising a nucleic acid molecule are well known in the art (see forexample EP 83 286; EP 206 920 for Copenhagen vaccinia viruses and Mayret al., 1975, Infection 3, 6-14; Sutter and Moss, 1992, Proc. Natl.Acad. Sci. USA 89, 10847-10851, U.S. Pat. No. 6,440,422 for MVAviruses). The nucleic acid molecule of the present disclosure istypically inserted within the poxyiral genome in a non-essential locus,such as non-coding intergenic regions or any gene for which inactivationor deletion does not significantly impair viral growth and replication.Thymidine kinase gene is particularly appropriate for insertion inCopenhagen vaccinia viruses (Hruby et al., 1983, Proc. Natl. Acad. Sci.USA 80, 3411-3415; Weir et al., 1983, J. Virol. 46, 530-537). As far asMVA is concerned, insertion of the nucleic acid molecule can beperformed in any of the excisions I to VII, and typically in excision Hor III (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038; Sutter et al.,1994, Vaccine 12, 1032-1040) or in D4R locus. For fowlpox virus,although insertion within the thymidine kinase gene may be considered,the nucleic acid molecule is typically introduced into a non-codingintergenic region (see for example EP 314 569 and U.S. Pat. No.5,180,675). One may also envisage insertion in an essential viral locusprovided that the defective function be supplied in trans, via a helpervirus or by expression in the producer cell line. Suitable poxyiralvectors can be readily generated from wild type poxviruses available inrecognized collections such as ATCC (fowlpox ATCC VR-251, monkey poxATCC VR-267, swine pox ATCC VR-363, canarypox ATCC VR-111, cowpox ATCCVR-302) or ICTV (Canberra, Australia) (Copenhagen virus code58.1.1.0.001; GenBank accession number M35027).

In certain embodiments, the vectors of the disclosure comprise thenucleic acid molecule of the disclosure in a form suitable for itsexpression in a host cell or organism, which means that the nucleic acidmolecule is placed under the control of one or more regulatorysequences, selected on the basis of the vector type and/or host cell,which is operatively linked to the nucleic acid molecule to beexpressed. As used herein, the term “regulatory sequence” refers to anysequence that allows, contributes or modulates the functional regulationof the nucleic acid molecule, including replication, duplication,transcription, splicing, translation, stability and/or transport of thenucleic acid or one of its derivative (i.e. mRNA) into the host cell ororganism. In the context of the disclosure, this term encompassespromoters, enhancers and other expression control elements (e.g.,polyadenylation signals and elements that affect mRNA stability).“Operably linked” is intended to mean that the nucleic acid molecule ofinterest is linked to the regulatory sequence(s) in a manner whichallows for expression of the nucleic acid molecule (e.g., in a host cellor organism). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, etc.

Regulatory sequences include promoters which direct constitutiveexpression of a nucleic acid molecule in many types of host cell andthose which direct expression of the nucleotide sequence only in certainhost cells (e.g., tissue-specific regulatory sequences) or in responseto specific events or exogenous factors (e.g. by temperature, nutrientadditive, hormone or other ligand).

Suitable regulatory sequences useful in the context of the presentdisclosure include, but are not limited to, the left promoter frombacteriophage lambda, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the cytomegalovirus (CMV) immediateearly promoter or enhancer (Boshart et al., 1985, Cell 41, 521-530), theadenovirus early and late promoters, the phosphoglycero kinase (PGK)promoter (Hitzeman et al., 1983, Science 219, 620-625; Adra et al.,1987, Gene 60, 65-74), the thymidine kinase (TK) promoter of herpessimplex virus (HSV)-1 and retroviral long-terminal repeats (e.g. MoMuLVand Rous sarcoma virus (RSV) LTRs). Suitable promoters useful to driveexpression of the nucleic acid molecule of the disclosure in a poxyiralvector include the 7.5K, H5R, TK, p28, p11 or K1L promoters of vacciniavirus. Alternatively, one may use a synthetic promoter such as thosedescribed in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097),Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar andBoyle (1990, Virology 179, 151-158) as well as chimeric promotersbetween early and late poxyiral promoters.

Inducible promoters are regulated by exogenously supplied compounds, andinclude, without limitation, the zinc-inducible metallothionein (MT)promoter (Mc Ivor et al., 1987, Mol. Cell. Biol. 7, 838-848), thedexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter,the T7 polymerase promoter system (WO 98/10088), the ecdysone insectpromoter (No et al., 1996, Proc. Natl. Acad. Sci. USA 93, 3346-3351),the tetracycline-repressible promoter (Gossen et al., 1992, Proc. Natl.Acad. Sci. USA 89, 5547-5551), the tetracycline-inducible promoter (Kimet al., 1995, J. Virol. 69, 2565-2573), the RU486-inducible promoter(Wang et al., 1997, Nat. Biotech. 15, 239-243 and Wang et al., 1997,Gene Ther. 4, 432-441) and the rapamycin-inducible promoter (Magari etal., 1997, J. Clin. Invest. 100, 2865-2872).

The regulatory sequences in use in the context of the present disclosurecan also be tissue-specific to drive expression of the nucleic acidmolecule in the tissues where therapeutic benefit is desired. Exemplaryliver-specific regulatory sequences include but are not limited to thoseof HMG-CoA reductase (Luskey, 1987, Mol. Cell. Biol. 7, 1881-1893);sterol regulatory element 1 (SRE-1; Smith et al., 1990, J. Biol. Chem.265, 2306-2310); albumin (Pinkert et al., 1987, Genes Dev. 1, 268-277);phosphoenol pyruvate carboxy kinase (PEPCK) (Eisenberger et al., 1992,Mol. Cell. Biol. 12, 1396-1403); human C-reactive protein (CRP) (Li etal., 1990, J. Biol. Chem. 265, 4136-4142); human glucokinase (Tanizawaet al., 1992, Mol. Endocrinology. 6, 1070-1081); cholesterol 7-alphahydroylase (CYP-7) (Lee et al., 1994, J. Biol. Chem. 269, 14681-14689);alpha-1 antitrypsin (Ciliberto et al., 1985, Cell 41, 531-540);insulin-like growth factor binding protein (IGFBP-1) (Babajko et al.,1993, Biochem Biophys. Res. Comm. 196, 480-486); human transferrin(Mendelzon et al., 1990, Nucl. Acids Res. 18, 5717-5721); collagen typeI (Houglum et al., 1994, J. Clin. Invest. 94, 808-814) and FIX (U.S.Pat. No. 5,814,716) genes. Exemplary prostate-specific regulatorysequences include but are not limited to those of the prostatic acidphosphatase (PAP) (Balms et al., 1994, Biochim. Biophys. Acta. 1217,188-194); prostatic secretory protein 94 (PSP 94) (Nolet et al., 1991,Biochim. Biophys. Acta 1089, 247-249); prostate specific antigen complex(Kasper et al., 1993, J. Steroid Biochem. Mol. Biol. 47, 127-135); humanglandular kallikrein (hgt-1) (Lilja et al., 1993, World J. Urology 11,188-191) genes. Exemplary pancreas-specific regulatory sequences includebut are not limited to those of pancreatitis associated protein promoter(Dusetti et al., 1993, J. Biol. Chem. 268, 14470-14475); elastase 1transcriptional enhancer (Kruse et al., 1993, Genes and Development 7,774-786); pancreas specific amylase and elastase enhancer/promoter (Wuet al., 1991, Mol. Cell. Biol. 11, 4423-4430; Keller et al., 1990, Genes& Dev. 4, 1316-1321); pancreatic cholesterol esterase gene promoter(Fontaine et al., 1991, Biochemistry 30, 7008-7014) and the insulin genepromoter (Edlund et al., 1985, Science 230, 912-916). Exemplaryneuron-specific regulatory sequences include but are not limited toneuron-specific enolase (NSE) (Forss-Petter et al., 1990, Neuron 5,187-197) and the neurofilament (Byrne and Ruddle, 1989, Proc. Natl.Acad. Sci. USA 86, 5473-5477) gene promoters. Exemplary regulatorysequences for expression in the brain include but are not limited to theneurofilament heavy chain (NF-H) promoter (Schwartz et al., 1994, J.Biol. Chem. 269, 13444-13450). Exemplary lymphoid-specific regulatorysequences include but are not limited to the human CGL1/granzyme Bpromoter (Hanson et al., 1991, J. Biol. Chem. 266, 24433-24438);terminal deoxy transferase (TdT), lymphocyte specific tyrosine proteinkinase (p561ck) promoters (Lo et al., 1991, Mol. Cell. Biol. 11,5229-5243); the human CD2 promoter/enhancer (Lake et al., 1990, EMBO J.9, 3129-3136), the human NK and T cell specific activation (NKG5)(Houchins et al., 1993, Immunogenetics 37, 102-107), T cell receptor(Winoto and Baltimore, 1989, EMBO J. 8, 729-733) and immunoglobulin(Banerji et al., 1983, Cell 33, 729-740; Queen and Baltimore, 1983, Cell33, 741-748) promoters. Exemplary colon-specific regulatory sequencesinclude but are not limited to pp 60c-src tyrosine kinase (Talamonti etal., 1993, J. Clin. Invest 91, 53-60); organ-specific neoantigens(OSNs), mw 40 kDa (p40) (Ilantzis et al., 1993, Microbiol. Immunol. 37,119-128); and colon specific antigen-P promoter (Sharkey et al., 1994,Cancer 73, 864-877) promoters. Exemplary regulatory sequences forexpression in mammary gland and breast cells include but are not limitedto the human alpha-lactalbumin (Thean et al., 1990, British J. Cancer.61, 773-775) and milk whey (U.S. Pat. No. 4,873,316) promoters.Exemplary muscle-specific regulatory sequences include but are notlimited to SM22 (WO 98/15575; WO 97/35974), the desmin (WO 96/26284),mitochondrial creatine kinase (MCK) promoters, and the chimeric promoterdisclosed in EP 1310561. Exemplary lung-specific regulatory sequencesinclude but are not limited to the CFTR and surfactant promoters.

Additional promoters suitable for use in this disclosure can be takenfrom genes that are preferentially expressed in proliferative tumorcells. Such genes can be identified for example by display andcomparative genomic hybridization (see for example U.S. Pat. Nos.5,759,776 and 5,776,683). Exemplary tumor specific promoters include butare not limited to the promoters of the MUC-1 gene overexpressed inbreast and prostate cancers (Chen et al., 1995, J. Clin. Invest. 96,2775-2782), of the Carcinoma Embryonic Antigen (CEA)-encoding geneoverexpressed in colon cancers (Schrewe et al., 1990, Mol. Cell. Biol.10, 2738-2748), of the ERB-2 encoding gene overexpressed in breast andpancreas cancers (Harris et al., 1994, Gene Therapy 1, 170-175), of thealpha-foetoprotein gene overexpressed in liver cancers (Kanai et al.,1997, Cancer Res. 57, 461-465), of the telomerase reverse transcriptase(TERT) (WO99/27113, WO 02/053760 and Horikawa et al., 1999, Cancer Res.59, 826), hypoxia-responsive element (HRE), autocrine motility factorreceptor, L plasmin and hexokinase II.

Those skilled in the art will appreciate that the regulatory elementscontrolling the expression of the nucleic acid molecule of thedisclosure may further comprise additional elements for properinitiation, regulation and/or termination of transcription andtranslation into the host cell or organism. Such additional elementsinclude but are not limited to non-coding exon/intron sequences,transport sequences, secretion signal sequences, nuclear localizationsignal sequences, IRES, polyA transcription termination sequences,tripartite leader sequences, sequences involved in replication orintegration. Illustrative examples of introns suitable in the context ofthe disclosure include those isolated from the genes encoding alpha orbeta globin (i.e. the second intron of the rabbit beta globin gene;Green et al., 1988, Nucleic Acids Res. 16, 369; Karasuyama et al., 1988,Eur. J. Immunol. 18, 97-104), ovalbumin, apolipoprotein, immunoglobulin,factor IX, and factor VIII, the SV40 16S/19S intron (Okayma and Berg,1983, Mol. Cell. Biol. 3, 280-289) as well as synthetic introns such asthe intron present in the pCI vector (Promega Corp, pCI mammalianexpression vector E1731) made of the human beta globin donor fused tothe mouse immunoglobin. Where secretion of the fusion protein isdesired, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the fusion protein orheterologous to both entities involved in the fusion protein. The personof ordinary skill in the art would be aware of the numerous regulatorysequences that are useful in expression vectors.

In addition, the vector of the disclosure can further comprise one ormore transgenes (i.e. a gene of interest to be expressed together withthe nucleic acid molecule of the disclosure in a host cell or organism).Desirably, the expression of the transgene has a therapeutic orprotective activity to the disease or illness condition for which thevector of the present disclosure is being given. Suitable transgenesinclude without limitation genes encoding (i) tumor proliferationinhibitors and/or (ii) at least one specific antigen against which animmune response is desired. In a typical form of the present disclosure,the transgene product and the fusion protein act synergistically in theinduction of immune responses or in providing a therapeutic (e.g.antitumoral) benefit. Accordingly, such combinations are not onlysuitable for immunoprophylaxis of diseases, but surprisingly also forimmunotherapy of diseases such as viral, bacterial or parasiticinfections, and also chronic disorders such as cancers.

Tumor proliferation inhibitors act by directly inhibiting cell growth,or killing the tumor cells. Representative examples of tumorproliferation inhibitors include toxins and suicide genes.Representative examples of toxins include without limitation ricin (Lambet al., 1985, Eur. J. Biochem. 148, 265-270), diphtheria toxin (Twetenet al., 1985, J. Biol. Chem. 260, 10392-10394), cholera toxin (Mekalanoset al., 1983, Nature 306, 551-557; Sanchez and Holmgren, 1989, Proc.Natl. Acad. Sci. USA 86, 481-485), gelonin (Stirpe et al., 1980, J.Biol. Chem. 255, 6947-6953), antiviral protein (Barbieri et al., 1982,Biochem. J. 203, 55-59; Irvin et al., 1980, Arch. Biochem. Biophys. 200,418-425), tritin, Shigella toxin (Calderwood et al., 1987, Proc. Natl.Acad. Sci. USA 84, 4364-4368; Jackson et al., 1987, Microb. Path. 2,147-153) and Pseudomonas exotoxin A (Carroll and Collier, 1987, J. Biol.Chem. 262, 8707-8711).

Specific antigens are typically those susceptible to confer an immuneresponse, specific and/or nonspecific, antibody and/or cell-mediated,against a given pathogen (virus, bacterium, fungus or parasite) oragainst a non-self antigen (e.g. a tumor-associated antigen). Typically,the selected antigen comprises an epitope that binds to, and ispresented onto the cell surface by MHC class I proteins. Representativeexamples of specific antigens include without limitation: antigen(s) ofthe Hepatitis B surface antigen are well known in the art and include,inter alia, those PreS1, Pars2 S antigens set forth described inEuropean Patent applications EP 414 374; EP 304 578, and EP 198 474.Antigens of the Hepatitis C virus including any immunogenic antigen orfragment thereof selected from the group consisting of the Core (C), theenvelope glycoprotein E1, E2, the non-structural polypeptide NS2, NS3,NS4 (NS4a and/or NS4b), NS5 (NS5a and/or NS5b) or any combinationthereof (e.g. NS3 and NS4, NS3 and NS4 and NS5b) Antigen(s) of the HIV-1virus, especially gp120 and gp160 (as described WO 87/06260). Antigen(s)derived from the Human Papilloma Virus (HPV) considered to be associatedwith genital warts (HPV 6 or HPV 11 and others), and cervical cancer(HPV16, HPV18, HPV 31, HPV-33 and others). Contemplated HPV antigens areselected among the group consisting of E5, E6, E7, L1, and L2 eitherindividually or in combination (see for example WO 94/00152, WO94/20137, WO 93/02184, WO 90/10459, and WO 92/16636). Contemplated inthe context of the disclosure are membrane anchored forms ofnon-oncogenic variants of the early HPV-16 E6 and/or E7 antigens (asdescribed in WO 99/03885) that are particularly suitable to achieve ananti-tumoral effect against an HPV-associated cancer. Antigens fromparasites that cause malaria. For example, typical antigens fromPlasmodia falciparum include RTS (WO 93/10152), and TRAP (WO 90/01496).Other plasmodia antigens that are likely candidates are P. falciparum.MSP1, AMA1, MSP3, EBA, GLURP, RAPT, RAP2, Sequestrin, PfEMP1, Pf332,LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27125, Pfs16,Pfs48/45, Pfs230 and their analogues in other Plasmodium species.

Other suitable antigens include tumour-associated antigens such as thoseassociated with prostrate, breast, colorectal, lung, pancreatic, renal,liver, bladder, sarcoma or melanoma cancers. Exemplary antigens includeMAGE 1, 3 and MAGE 4 or other MAGE antigens (WO 99/40188), PRAME, BAGE,Lage (also known as NY Eos 1) SAGE and HAGE (WO 99/53061) or GAGE(Robbins and Kawakami, 1996. Current Opinions in Immunol. 8, pps628-636). Other suitable tumor-associated antigens include those knownas prostase, including Prostate specific antigen (PSA), PAP, PSCA, PSMA.Prostase nucleotide sequence and deduced polypeptide sequence andhomologs are disclosed in Ferguson, et al. (1999, Proc. Natl. Acad. Sci.USA. 96, 3114-3119) and WO 98/12302 WO 98/20117 and WO 00/04149. Othersuitable tumour-associated antigens include those associated with breastcancer, such as BRCA-1, BRCA-2 and MUC-1 (see for example WO 92/07000).

The transgene in use in the present disclosure is placed under thecontrol of appropriate regulatory elements to permit its expression inthe selected host cell or organism in either a constitutive or induciblefashion. The choice of such regulatory elements is within the reach ofthe skilled artisan. It is typically selected from the group consistingof constitutive, inducible, tumor-specific and tissue-specific promotersas described above in connection with the expression of the fusionprotein of the present disclosure. In one example, the transgene isplaced under control of the CMV promoter to ensure high levelexpression.

The transgene in use in the present disclosure can be inserted in anylocation of the vector. According to one alternative, it is placedtypically not in close proximity of the nucleic acid molecule of thedisclosure. According to another alternative it can be placed inantisense orientation with respect to the nucleic acid molecule, inorder to avoid transcriptional interference between the two expressioncassettes. For example, in an adenoviral genome, the transgene can beinserted in a different deleted region with respect to the nucleic acidmolecule of the disclosure (E1, E3 and/or E4) or in the same deletedregion as said nucleic acid molecule but in antisense orientation to oneanother.

Introducing the nucleic acid molecule of the disclosure into a vectorbackbone can proceed by any genetic engineering strategy appropriate inthe art for any kind of vectors such as by methods described in Sambrooket al. (2001, Molecular Cloning—A Laboratory Manual, Cold Spring HarborLaboratory). Typically, for the introduction of the nucleic acidmolecule into an adenoviral vector, a bacterial plasmid comprising thefusion-encoding nucleic acid molecule is engineered to replace anadenoviral gene required for replication or assembly (e.g. E1) with thesubstitute nucleic acid molecule. The plasmid is then used as a shuttlevector, and combined with a second plasmid containing the complementaryportion of the adenovirus genome, permitting homologous recombination tooccur by virtue of overlapping adenovirus sequences in the two plasmids.The recombination can be done directly in a suitable mammalian host(such as 293 as described in Graham and Prevect, 1991, Methods inMolecular Biology, Vol 7“Gene Transfer and Expression Protocols”; Ed E.J. Murray, The Human Press Inc, Clinton, N.J.), or else in yeast YACclones or E. coli (as described in WO 96/17070). The completedadenovirus genome is subsequently transfected into mammalian host cellsfor replication and viral encapsidation.

The present disclosure also encompasses vectors of the disclosure orparticles thereof that have been modified to allow preferentialtargeting of a particular target cell. A characteristic feature oftargeted vectors/particles of the disclosure (of both viral andnon-viral origins, such as and lipid-complexed vectors) is the presenceat their surface of a targeting moiety capable of recognizing andbinding to a cellular and surface-exposed component. Such targetingmoieties include without limitation chemical conjugates, lipids,glycolipids, hormones, sugars, polymers (e.g. PEG, polylysine, PEI andthe like), peptides, polypeptides (for example JTS1 as described in WO94/40958), oligonucleotides, vitamins, antigens, lectins, antibodies andfragments thereof. They are typically capable of recognizing and bindingto cell-specific markers, tissue-specific markers, cellular receptors,viral antigens, antigenic epitopes or tumor-associated markers. In thisregard, cell targeting of adenoviruses can be carried out by geneticmodification of the viral gene encoding the capsid polypeptide presenton the surface of the virus (e.g. fiber, penton and/or pIX). Examples ofsuch modifications are described in literature (for example in Wickam etal., 1997, J. Viral. 71, 8221-8229; Amberg et al., 1997, Virol. 227,239-244; Michael et al., 1995, Gene Therapy 2, 660-668; WO 94/10323, EP02 360204 and WO 02/96939). To illustrate, inserting a sequence codingfor EGF within the sequence encoding the adenoviral fiber will allow totarget EGF receptor expressing cells. The modification of poxyiraltropism can also be achieved as described in EP 1 146 125. Other methodsfor cell specific targeting can be achieved by the chemical conjugationof targeting moieties at the surface of a viral particle.

In certain embodiments, the present disclosure relates to infectiousviral particles comprising the above-described nucleic acid molecules orvectors of the present disclosure.

The disclosure also relates to a process for producing an infectiousviral particle, comprising the steps of: (a) introducing the viralvector of the disclosure into a suitable cell line, (b) culturing saidcell line under suitable conditions so as to allow the production ofsaid infectious viral particle, and (c) recovering the producedinfectious viral particle from the culture of said cell line, and (d)optionally purifying said recovered infectious viral particle.

The vector containing the nucleic acid molecule of the disclosure can beintroduced into an appropriate cell line for propagation or expressionusing well-known techniques readily available to the person of ordinaryskill in the art. These include, but are not limited to, microinjectionof minute amounts of DNA into the nucleus of a cell (Capechi et al.,1980, Cell 22, 479-488), CaPO.sub.4-mediated transfection (Chen andOkayama, 1987, Mol. Cell Biol. 7, 2745-2752), DEAE-dextran-mediatedtransfection, electroporation (Chu et al., 1987, Nucleic Acid Res. 15,1311-1326), lipofection/liposome fusion (Feigner et al., 1987, Proc.Natl. Acad. Sci. USA 84, 7413-7417), particle bombardment (Yang et al.,1990, Proc. Natl. Acad. Sci. USA 87, 9568-9572), gene guns,transduction, infection (e.g. with an infective viral particle), andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).

When the vector of the disclosure is defective, the infectious particlesare usually produced in a complementation cell line or via the use of ahelper virus, which supplies in trans the non-functional viral genes.For example, suitable cell lines for complementing adenoviral vectorsinclude the 293 cells (Graham et al., 1997, J. Gen. Virol. 36, 59-72) aswell as the PER-C6 cells (Fallaux et al., 1998, Human Gene Ther. 9,1909-1917) commonly used to complement the E1 function. Other cell lineshave been engineered to complement doubly defective adenoviral vectors(Yeh et al., 1996, J. Virol. 70, 559-565; Krougliak and Graham, 1995,Human Gene Ther. 6, 1575-1586; Wang et al., 1995, Gene Ther. 2, 775-783;Lusky et al., 1998, J. Virol. 72, 2022-2033; WO94/28152 and WO97/04119).The infectious viral particles may be recovered from the culturesupernatant but also from the cells after lysis and optionally arefurther purified according to standard techniques (chromatography,ultracentrifugation in a cesium chloride gradient as described forexample in WO 96/27677, WO 98/00524, WO 98/22588, WO 98/26048, WO00/40702, EP 1016700 and WO 00/50573).

The disclosure also relates to host cells which comprise the nucleicacid molecules, vectors or infectious viral particles of the disclosuredescribed herein. For the purpose of the disclosure, the term “hostcell” should be understood broadly without any limitation concerningparticular organization in tissue, organ, or isolated cells. Such cellsmay be of a unique type of cells or a group of different types of cellsand encompass cultured cell lines, primary cells and proliferativecells.

Host cells therefore include prokaryotic cells, lower eukaryotic cellssuch as yeast, and other eukaryotic cells such as insect cells, plantand higher eukaryotic cells, such as vertebrate cells and, with aspecial preference, mammalian (e.g. human or non-human) cells. Suitablemammalian cells include but are not limited to hematopoietic cells(totipotent, stem cells, leukocytes, lymphocytes, monocytes,macrophages, APC, dendritic cells, non-human cells and the like),pulmonary cells, tracheal cells, hepatic cells, epithelial cells,endothelial cells, muscle cells (e.g. skeletal muscle, cardiac muscle orsmooth muscle) or fibroblasts. Typical host cells include Escherichiacoli, Bacillus, Listeria, Saccharomyces, BHK (baby hamster kidney)cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells(Crandell feline kidney cell line), CV-1 cells (African monkey kidneycell line), COS (e.g., COS-7) cells, chinese hamster ovary (CHO) cells,mouse NIH/3T3 cells, HeLa cells and Vero cells. Host cells alsoencompass complementing cells capable of complementing at least onedefective function of a replication-defective vector of the disclosure(e.g. adenoviral vector) such as those cited above.

The host cell of the disclosure can contain more than one nucleic acidmolecule, vector or infectious viral particle of the disclosure. Furtherit can additionally comprise a vector encoding a transgene, e.g. atransgene as described above. When more than one nucleic acid molecule,vector or infectious viral particle is introduced into a cell, thenucleic acid molecules, vectors or infectious viral particles can beintroduced independently or co-introduced.

Moreover, according to a specific embodiment, the host cell of thedisclosure can be further encapsulated. Cell encapsulation technologyhas been previously described (Tresco et al., 1992, ASAJO J. 38, 17-23;Aebischer et al., 1996, Human Gene Ther. 7, 851-860). According to saidspecific embodiment, transfected or infected eukaryotic host cells areencapsulated with compounds which form a microporous membrane and saidencapsulated cells can further be implanted in vivo. Capsules containingthe cells of interest may be prepared employing hollow microporousmembranes (e.g. Akzo Nobel Faser A G, Wuppertal, Germany; Deglon et al.1996, Human Gene Ther. 7, 2135-2146) having a molecular weight cutoffappropriate to permit the free passage of proteins and nutrients betweenthe capsule interior and exterior, while preventing the contact oftransplanted cells with host cells.

Still a further aspect of the present disclosure is a method forrecombinantly producing the fusion protein, employing the vectors,infectious viral particles and/or host cells of the disclosure. Themethod for producing the fusion protein comprises introducing a vectoror an infectious viral particle of the disclosure into a suitable hostcell to produce a transfected or infected host cell, culturing in-vitrosaid transfected or infected host cell under conditions suitable forgrowth of the host cell, and thereafter recovering said fusion proteinfrom said culture, and optionally, purifying said recovered fusionprotein. It is expected that those skilled in the art are knowledgeablein the numerous expression systems available for expression of thefusion proteins of the disclosure in appropriate host cells.

The host cell of the disclosure is typically produced bytransfecting/infecting a host cell with one or more recombinantmolecules, (e.g. a vector of the disclosure) comprising one or morenucleic acid molecules of the present disclosure. Recombinant DNAtechnologies can be used to improve expression of the nucleic acidmolecule in the host cell by manipulating, for example, the number ofcopies of the nucleic acid molecule within a host cell, the efficiencywith which the nucleic acid molecule is transcribed, the efficiency withwhich the resultant transcripts are translated, the efficiency ofpost-translational modifications and the use of appropriate selection.Recombinant techniques useful for increasing the expression of nucleicacid molecules of the present disclosure include, but are not limitedto, the use of high-copy number vectors, addition of vector stabilitysequences, substitution or modification of one or more transcriptionalregulatory sequences (e.g., promoters, operators, enhancers),substitution or modification of translational regulatory sequences(e.g., ribosome binding sites, Shine-Dalgamo sequences), modification ofnucleic acid molecule of the present disclosure to correspond to thecodon usage of the host cell, and deletion of sequences that destabilizetranscripts.

Host cells of the present disclosure can be cultured in conventionalfermentation bioreactors, flasks, and petri plates. Culturing can becarried out at a temperature, pH and oxygen content appropriate for agiven host cell. No attempts to describe in detail the various methodsknown for the expression of proteins in prokaryote and eukaryote cellswill be made here. In one embodiment, the vector is a plasmid carryingthe fusion-encoding nucleic acid molecule in operative association withappropriate regulatory elements. Typical host cells in use in the methodof the disclosure are mammalian cell lines, yeast cells and bacterialcells.

Where the fusion protein is not secreted outside the producing cell orwhere it is not secreted completely, it can be recovered from the cellby standard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. If secreted,it can be recovered directly from the culture medium. The fusion proteincan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, gelelectrophoresis, reverse phase chromatography, size exclusionchromatography, ion exchange chromatography, affinity chromatography,phosphocellulose chromatography, hydrophobic-interaction chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography. The conditions and technology used topurify a particular fusion protein of the disclosure will depend on thesynthesis method and on factors such as net charge, molecular weight,hydrophobicity, hydrophilicity and will be apparent to those havingskill in the art. It is also understood that depending upon the hostcell used for the recombinant production of the fusion proteinsdescribed herein, the fusion proteins can have various glycosylationpatterns, or may be non-glycosylated (e.g. when produced in bacteria).In addition, the fusion protein may include an initial methionine insome cases as a result of a host-mediated process.

The fusion protein of the disclosure can be “purified” to the extentthat it is substantially free of cellular material. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the fusionprotein, even if in the presence of considerable amounts of othercomponents. In some uses, “substantially free of cellular material”includes preparations of the fusion protein having less than about 30%(by dry weight) other proteins (i.e., contaminating proteins), typicallyless than about 20% other proteins, more typically less than about 10%other proteins, or even more typically less than about 5% otherproteins. When the fusion protein is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

Terms

As used herein, the term “conjugate” refers to molecular entities joinedby covalent bonds or other arrangement that provides substantiallyirreversible binding under physiological conditions. For example, twoproteins, isolated and/or purified polypeptide sequence, may beconjugated together by a linker polymer, e.g., amino acid, polypeptidesequence, ethylene glycol polymer. Two proteins may be conjugatedtogether by linking one protein to a ligand and linking the secondprotein to a receptor, e.g., streptavidin and biotin or an antibody andan epitope.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, “subject” refers to any animal, typically a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, “amino acid sequence” refers to an amino acid sequenceof a protein molecule. An “amino acid sequence” can be deduced from thenucleic acid sequence encoding the protein. However, terms such as“polypeptide” or “protein” are not meant to limit the amino acidsequence to the deduced amino acid sequence, but include non-naturallyoccurring amino acids, post-translational modifications of the deducedamino acid sequences, such as amino acid deletions, additions, andmodifications such as glycolsylations and addition of lipid moieties.

The term “a nucleic acid sequence encoding” a specified polypeptiderefers to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a geneproduct. The coding region may be present in a cDNA, genomic DNA or RNAform. When present in a DNA form, the oligonucleotide, polynucleotide,or nucleic acid may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present disclosure may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

A “virus-like particle” refers to a particle comprising virion proteinsbut is substantially free of viral genetic material, e.g., viral RNA.Virus-like particles may contain viral proteins from different viruses.See e.g., Guo et al., Enhancement of mucosal immune responses bychimeric influenza HA/SHIV virus-like particles, Virology, 2003,313(2):502-13. Virus-like particles may contain lipid membranes and maybe constructed to express a variety of antigens on their particlesurface ether by expression in viral vectors use to create the particlesor by mixing the virus-like particle with an antigen or otherpolypeptide conjugated to a glycosylphosphatidyl-inositol anchor. Seee.g. Skountzou et al., J. Virol. 81(3):1083-94; Derdak et al., PNAS,2006, 103(35) 13144-13149; Poloso et al., Molecular Immunology, 2001,38:803-816.

As used herein, the article “a” or “an” is intended to refer to one ormore unless the context suggests otherwise.

Experimental

GIFT4 Gene and Protein.

The genes (cDNA) of murine IL-4 and GM-CSF were purchased from Invivogen(San-Diego, Calif.), and cloned into the bicistronic AP2 retrovector ina frame allowing the expression of both the chimeric transgenes andGIFT4 fusion proteins. One amino acid (Serine, S) serves as the bridgelinker between the GM-CSF and IL-4 protein sequences. To build thethree-dimensional structure of murine GIFT4 protein, the crystalstructures of human GM-CSF and IL-4 were used as the templates forhomology modeling on the software PROSPECT v2 (Oak Ridge NationalLaboratory, Oak Ridge, Tenn.). The GIFT4-encoding retroviral plasmid wasintroduced into the 293-GP2 packaging cells (Clontech, Mountain View,Calif.) following the manufacturer's instructions. The concentratedretroparticles encoding GIFT4 or GM-CSF or IL-4 genes were used togenetically modify 293T cells or B16F10 melanoma cells. 293T-GIFT4 cellsor B16F0-GIFT4 cells were pooled together from positive single cellclone selections in the wells of a 96-well plate, confirmed by GIFT4protein expression detected by ELISA.

GIFT4 Triggers B Cell Expansion.

In order to test the immune stimulatory function of GIFT4 fusokine,murine GIFT4 cDNA from parental GM-CSF and IL-4 cDNAs was cloned into anAP2 retrovirus vector, then transfected into 293T cells. The translatedGIFT4 protein sequence is consistent of a single polypeptide chain of282 amino acids (FIG. 1A) with a predicted 3D structure (FIG. 1B).AP2-GIFT4 vector-transfected 293T cells stably express abundant GIFT4protein with a about 50 kDa of molecular weight (FIG. 1C). GIFT4 fusionprotein has strong bioactivities to induce proliferation ofGM-CSF-responder JAWSII cells (FIG. 1D) and IL-4-dependent CT.h4S cells(FIG. 1E).

To examine the immune function of GIFT4 protein, splenocytes wereisolate from C57BL/6J (B6) mice, and stimulate the cells with GIFT4protein in comparison with combined use of its parental molecules GM-CSFand IL-4 in vitro. GIFT4 triggers the expansion of splenocytes (FIG.9A), unexpectedly in the B-cell compartment (FIG. 9B). Consistently,GIFT4 induce the proliferation of purified B cells (FIG. 2A-B). Todefine the phenotype of GIFT4-treated splenic B cells (GIFT4-B cells),we profiled GIFT4-B cells with a panel of surface markers. FACS analysesdemonstrated that GIFT4-B cells express B220, CD19, CD22, CD25, CD40,MHCI/II, IgM, CD80 and CD86 (FIG. 2C); the latter two are the commonmarkers for antigen-presenting cells. BCR cross-linking with anti-murineIgM further confirmed that GIFT4-B cells have the plasticity to switchthe isotype of immunoglobulin expression on the cells from IgM to IgG(FIG. 2D), with down-regulation of CD80 and CD86.

GIFT4 Triggers Anti-Tumor Immunity.

Cytokine secretion by B cells plays important roles in both innate andadaptive immunities against infectious pathogens and tumors. IL-4 is a γchain family member that induces the phosphorylation of STAT6. To testthe capacity of GIFT4 on STAT signaling, purified murine B cells werestimulated with GIFT4 protein, compared to the stimulation of individualor combined recombinant GM-SCF and IL-4. GIFT4 possessesgain-of-function on the phosphorylation of STAT1, STAT3, STAT5 and STAT6(FIG. 3A). To check cytokine production by GIFT4-B cells, the culturesupernatant of purified splenic B cell stimulated with GIFT4 protein wassubject to cytokine luminex analyses. The prolife of secretome revealedthat GIFT4-B cells produce IL-1β, IL-6, IL-12 (FIG. 3B), IL-5, VEGF, andmassive amount of GM-CSF and the chemokine CCL3 (FIG. 3C), withundetectable IL-10 and little IFN-γ (FIG. 3B), as well as other lowerlevel of cytokines. Intracellular cytokine staining further confirmedthe secretion of GM-CSF by GIFT4-B cells (FIG. 3D), which is more thanten folds higher in comparison with the one in control treatment withcombined use of recombinant GM-CSF and IL-4 (FIG. 3E). To test theeffect of GIFT4 protein on GM-CSF-producing B cells in vivo, GIFT4protein was administrated into B5 mice by intravenous injection. Afterone week of GIFT4 treatment, the mice developed splenomegaly (FIG. 4A);mice treated with combined GM-CSF and IL-4 showed normal size of spleens(FIG. 4A) as untreated mice (not shown). Profiling B220⁺ cells and CD3⁺cells in the spleens from those mice by FACS demonstrated that there wasrobust expansion of splenic B cells in GIFT4-treated mice, compared withthe mice treated with GM-CSF and IL-4 (FIG. 4B); there was also slight Tcell proliferation in GIFT4-treated mice. Intracellular staining furtherconfirmed the induction of GM-CSF-secreting splenic B cells by GIFT4treatment. The percentage of GM-CSF⁺ B cells in GIFT4-treated mice ismore than 30 folds higher than the one in mice treated with GM-CSF andIL-4 (FIG. 10B), the latter is similar to normal untreated mice.

GM-CSF is contemplated as a cytokine adjuvant for tumor vaccine. GIFT4-Bcells secrete IL12, IL-6 and IL-1β, which could enhance Th1 T cellresponse and the production of IFN-γ that is an essential anti-tumorcytokines. Therefore, GIFT4 protein could elicit anti-tumor immunity invivo. To test, melanoma mouse model was established by subcutaneousimplantation of B16F0 melanoma cells into B6 mice. Five days after tumorimplant, there were visible tumors developed in the mice. Those micewere treated with GIFT4 protein or combined GM-CSF and IL-4, or PBS asuntreated control. Two weeks later, the mice in control group orcombined cytokine treatment developed massive melanoma tumors (FIG. 4C);in contrast, GIFT4 treatment significantly suppressed tumor growth (FIG.4C).

To further test the anti-tumor function of GIFT4 protein, geneticallymodified B16F0 melanoma cell line stably expressing GIFT4 protein(B16F0-GIFT4 cells) or individual GM-CSF or IL-4 cytokine weregenerated. The tumor cells were injected subcutaneously into syngeneicB6 mice. Twenty days later, mice implanted with wild type B16F0 cells orwith mixed B16F0-GMCSF and B 16F0-IL4 cells (B16F0-GMCSF+IL4) developedsubstantial melanoma tumors (FIG. 4D); however, tumor growth wasdramatically inhibited in mice implanted with B 16F0-GIFT4 cells,indicating GIFT4 expression significantly suppressed melanoma tumorgrowth (FIG. 4D).

GIFT4-Elicited Anti-Tumor Immunity is B-Cell Dependent.

Anti-tumor immunity consistent of two arms of innate and adaptive immunecompartments. To check whether GIFT4 protein targets on the adaptivearm, B16F0-GIFT4 cells were implanted in Rag1^(−/−) mice that lackfunctional B cells and T cells. Melanoma tumor grew quickly inRag1^(−/−) mice (FIG. 5A). T cells play roles in anti-tumor immunity.Growth of B16F0-GIFT4 tumors in CD4 T cell or CD8 T cell deficient micewas observed (FIG. 5B). To further test whether B cells play a pivotalrole in GIFT4-triggered anti-tumor response, B16F0-GIFT4 cells wereimplanted into B cell deficient μMT mice. Consistent with the immunefunction of GIFT4 protein on B cells in vitro and in vivo, μMT mice thatexclusively lack functional B cells developed large size of melanomatumors (FIG. 5B). GIFT4-B cells secretes IL-12, IL-6 and IL-1β which canenhance IFN-γ production by T cells. To test whether GIFT4-B cells couldinteract T cells and promote anti-tumor immunity the hypothesis, T cellswere co-cultured with purified B cells stimulated with GIFT4 protein, orindividual GM-CSF or IL-4, or combined recombinant cytokines.Quantification of IFN-γ secretion in the culture supernatant by ELISAshowed that GIFT4 stimulation robustly increased IFN-γ production by Tcells (FIG. 5C), while control treatments with individual recombinantcytokine GM-CSF, IL-4 or combined use had no significant effect on Tcell IFN-γ production. Using gene knockout mice, it was confirmed thatmice deficient with IL-12 or IFN-γ, but not IL-10, could not suppressmelanoma tumor growth (FIG. 5D).

Tumor-Specific Antibody is Important for GIFT4-Triggered Anti-TumorImmunity.

B cell immunity includes cellular and humoral immune responses. To testwhether GIFT4 could boost B cell antibody response, normal B6 mice wereimmunized with ovalbumin (OVA) in presence of GIFT4 protein or mixedGM-CSF and IL-4. Mice injected with control medium absent OVA served asthe control (FIG. 6A). ELISpot analyses of OVA-specific IgG-secreting Bcells from the harvested spleens demonstrated that OVA with GIFT4treatment significantly enhanced antigen-specific antibody production invivo, compared with OVA plus GM-CSF and IL-4 (FIG. 6B). There wasundetectable OVA-specific IgG secreting B cells existed in the spleenwhen no antigen was administrated in the control mice. To examine thecapability of GIFT4 protein as an adjuvant to boost anti-tumor specificantibodies, B6 or μMT B-cell deficient mice were immunized with B16F0-GIFT4 melanoma cells. One month later, serum was collected from themice. Flow cytometry analyses of B16F0 cells treated with the serum fromthe immunized mice or naïve B6 mice verified the presence of high titleof anti-melanoma specific antibodies in immunized B6 mice (FIG. 6C-D).B-cell deficient mice μMT mice and naïve B6 mice have undetectableanti-B16F0 antibodies in the circulation. To test whether theanti-melanoma antibodies involved in GIFT4-triggered B cell-mediatedanti-tumor immunity, B16F0-GIFT4 melanoma cells were subcutaneouslyimplanted into B6, FcγR^(−/−) or μMT mice. FcγR^(−/−) mice lackfunctional IgG. Monitoring tumor growth demonstrated that there wasmassive tumor growth in FcγR^(−/−) mice, as well as in μMT B-celldeficient mice, but not in the wild type mice (FIG. 6E). To examinewhether the immunization of B16F0-GIFT4 cells could elicit protectiveimmunity against melanoma, immunized or unimmunized mice were challengedwith B16F0 tumor cells. Immunization of B16-GIFT4 cells completelyprevented the mice from developing melanoma tumors (FIG. 7A). Incontrast, mice without immunization developed large tumors (FIG. 7A).Adoptive transfer of immune cells is a promising approach for cancercell immunotherapy. To further investigate whether GIFT4-activated Bcells from immunized mice could pass the active anti-tumor immunity intoB cell deficient mice, B16F0-GIFT4 cells were implanted into μMT mice.When the mice developed a visible size of melanoma tumors, splenic Bcells purified from immunized mice were adoptively transferred into themice. Measurement of the tumor size showed that adoptive transfer ofB16F0-primed B cells from immunized mice significantly inhibitedmelanoma tumor growth in B-cell deficient μMT mice (FIG. 7B). Micewithout B-cell adoptive transfer developed large size of melanomatumors.

Human GM-CSF and IL-4 Derived Fusion Cytokine Reprograms LeukemicB-Cells to Anti-CLL Effectors

A human GM-CSF and IL-4 derived fusokine GIFT4 was generated to test itsimmune function on chronic lymphoid leukemia B-cells (CLL-B cells).(FIG. 11) Human GIFT4 protein reprograms leukemic B cells into anti-CLLeffectors and helpers. (FIG. 10) GIFT4 activated CLL B cells viaexclusive hyper-phosphorylation of STATS. Unlike the induced expansionof normal human B cells, GIFT4 did not trigger CLL B-cell proliferation.(FIG. 12) GIFT4-converted CLL B-cells up-regulated the expression ofco-stimulatory molecules CD40, CD80 and CD86, behaved likeantigen-presenting cells, and secreted IL-1β, IL-6, ICAM1 and massiveIL-2. (FIG. 13) GIFT4-CLL B cells further propelled the expansion ofIFN-γ-producing autologous cytotoxic NK and T cells. Co-cultureGIFT4-treated CLL cells significantly increased the killing of primaryautologous CLL cells ex vivo. (FIG. 14) Together, these data demonstratethat GIFT4 has potent anti-CLL immune function by reprograming leukemicB cells into anti-CLL helper cells. Fusokine GIFT4 protein andGIFT4-converted CLL-B cells could serve as novel immunotherapeutic forCLL treatment.

Cell Culture

GIFT4-secreting 293T cells or B16F0 melanoma cell line, ornon-transfected cells were cultured in DMEM medium (Wisent Technologies,Rocklin, Calif.) supplemented with 10% FBS (Wisent Technologies) and 50U/ml of Pen/Strep antibiotics (Wisent Technologies). Culture supernatantwas collected and concentrated with sterile centrifugal filter units(Millipore Corporation, Billerica, Mass.) for ELISA assay and Westernblot. The concentrated culture supernatant of 293T-GIFT4 cells wasfurther used for in vitro and in vivo experiments. Splenocytes fromC57BL/6J mice or B cells (10⁵ cells/well) purified from splenocytes bynegative selection with B-cell enrichment kit (StemCell, Montreal,Canada) were cultured in complete RPMI 1640 medium for 6 days inpresence of 2 ng/ml of GIFT4 protein or recombinant GM-CSF and IL-4control proteins (R&D system, Minneapolis, USA). Alternatively, B cellswere labeled with CFSE dye (Invitrogen, Eugene, Oreg.) and cultured incomplete RPMI 1640 medium for cell proliferation assay following theinstruction from the company.

ELISA and Western Blot

Quantification of GIFT4 protein expressed by transfused 293T or B16F0melanoma tumor cells was performed with ELISA kits for murine GM-SCF orIL-4 (eBiosciences, San Diego, Calif.) following the instruction fromthe company. IFN-γ production by T lymphocytes in vitro was determinedwith IFN-γ ELISA kit from eBiosciences. Intact murine GIFT4 proteinswere analyzed by Western blot with anti-mouse MG-SCF or anti-IL-4specific antibodies (R&D systems). STAT phosphorylation activated byGIFT4 stimulation in B cells was profiled by Western blot withanti-pSTAT1, anti-pSTAT3, anti-pSTAT5, anti-pSTAT6, or anti-STATantibodies (Cell Signaling, Boston, Mass.).

MTT Assay

For determining the bioactivity of IL-4 or GM-CSF fusion compartments ofGIFT4 protein, IL-4-responsive CT.h4S cells (Provided by the laboratoryof Dr. William Paul in National Institutes of Health, USA) andGM-CSF-responsive JASWII cells were plated at a density of 5,000 cellsper well in a 96-well plate, and cultured in complete RPMI 1640 mediumsupplemented with 2 ng/ml of recombinant IL-4 cytokines or 10 ng/ml ofrecombinant GM-CSF (PeproTech, Rocky Hill, N.J.) respectively, or withGIFT4 protein (2 ng/ml for CT.h4S cells and 10 ng/ml for JASWII cells).

After 72-hour culture, 20 μL of3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)(Sigma, Saint Louis, Mo.) solution was added for 4 hours of incubationat 37° C. Cell pellet was dissolved in 200 μL of absolute DMSO (QualityBiological Inc., Solon, Ohio), and read at an absorbance of 570 nm on amicroplate spectrophotometer (BioTek Instruments Inc., Winooski, Vt.).

Cell Flow Cytometry

GIFT4-treated splenocytes in vitro were stained with APC-conjugatedanti-mouse B220 and PE-conjugated anti-mouse CD3 antibodies. B-cell andT-cell profiles were analyzed by cell flow cytometry (FACS) on a BDFACSCanto II flow cytometer. Surface markers of GIFT4-treated B cellspurified from splenocytes were profiled by flow cytometry with a panelof B cell surface markers (anti-B220, CD19, CD22, CD23, CD25, CD27,CD40, CD69, CD80, CD86, MHCI, MHCII, IgM, IgG) (BD, San Diego, Calif.).For intracellular GM-CSF staining, B cells were fixed and permeabilizedwith BD Cytofix/Cytoperm™ solution followed by GM-CSF antibody staining.Alternatively, murine GIFT4 protein (20 ng) was intravenously injectedinto C57BL/6J mice at day 0, 2 and 4. Recombinant murine GM-CSF and IL-4(20 ng) served as protein control. Splenocytes were isolated fromspleens of treated mice on day 6. Total B cells and T cells wereprofiled by cell flow cytometry with anti-B220 and anti-CD3 antibodies(BD), and the cell number per spleen was calculated. The production ofspecific antibodies against B16F0 melanoma cells in vivo was examined byFACS by incubation of serum from immunized or control mice with themelanoma cells, following with the staining of APC-conjugated donkeyanti-mouse secondary antibodies (BD). FACS data were analyzed withFlowJo 9.1 software.

B Cell ELISpot

C57BL/6J mice were administrated by intraperitoneal injection with OVAprotein (10 μg/mouse/time) supplemented with GIFT4 protein (20ng/mouse/time) or combined recombinant GM-CSF and IL-4 (20ng/mouse/time) on day 0 and 7. Mice without cytokine treatment served asblank control (n=5 in each group). On day 14, spleens were harvested,and B cells were purified from splenocytes by negative selection withB-cell enrichment kit (StemCell). The number of OVA-specificIgG-secreting cells per 50,000 B cells was analyzed by B-cell ELISpotkit (Mabtech, Cincinnati, Ohio) following the instruction provided bythe manufacturer.

Luminex Assay

The culture supernatants of GIFT4-treated B cells were collected on day5, and subject to luminex assay with murine 26-plex cytokine polystyrenebead kit (Affymetrix, Santa Clara, Calif.) performed in Human ImmunologyMonitoring Center of Stanford University, according to themanufacturer's instruction. Samples were read on a Luminex 200instrument with a lower bound of 100 beads per sample per cytokine.

Murine Melanoma Model

B 16F0 or GIFT4-producing B 16F0 melanoma cells (10⁶/mouse) weresubcutaneously implanted into syngeneic C57BL/6J mice or Rag1^(−/−),CD4^(−/−), CD8^(−/−), μMT (B-cell deficient) or FcγR^(−/−) (IgGfunction-deficient) mice. Alternatively, GIFT4-secreting B 16F0 cells(10⁶ cells/mouse) were subcutaneously immunized into C57BL/6J mice.After 30 days, wild type B16F0 melanoma cells (10⁶/mouse) were implantedinto the immunized mice. Unimmunized mice served as controls.Additionally, 10 millions of splenocytes or 5 millions of purified Bcells isolated from immunized mice or control mice were adoptivelytransferred by intravenous injection into C57BL/6J mice withpre-established B16F0 melanoma. For testing the anti-tumor function ofGIFT4 protein, C57BL/6J mice with pre-established B 16F0 tumors wereadministrated with three doses of 100 ng/day/mouse of murine GIFT4 with2 days interval. In addition, purified splenic B cells (10×10⁶cells/mouse) from immunized mice were adoptively transferred into B16F0tumor pre-established μMT mice; mice without cell adoptive transferserved as control. Tumor growth was measured with a digital caliper.Mice used are female (6-8 weeks old) purchased from Jackson Laboratory(Bar Harbor, Me.).

The invention claimed is:
 1. A method of treating cancer comprisingadministering a conjugate comprising the amino acid sequence of SEQ IDNO: 8 to a subject in need thereof, wherein the cancer is ahematological malignancy.
 2. The method of claim 1, wherein thehematological malignancy is selected from leukemia, lymphoma, acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronicmyelogenous leukemia, acute monocytic leukemia (AMOL), Hodgkin'slymphomas, and non-Hodgkin's lymphomas such as Burkitt lymphoma, B-celllymphoma and multiple myeloma.
 3. A method of treating melanomacomprising administering a conjugate comprising the amino acid sequenceof SEQ ID NO: 8 to a subject in need thereof.